Method for making a biofabricated material containing collagen fibrils

ABSTRACT

Described herein is a method for producing a biofabricated material from collagen or collagen-like proteins. The collagen or collagen-like proteins are isolated from animal sources or produced by recombinant DNA techniques or by chemical synthesis. The collagen or collagen-like proteins are fibrillated, crosslinked, dehydrated and lubricated thus forming the biofabricated material having a substantially uniform network of collagen fibrils.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/295,435 filed Feb. 15, 2016 and which is incorporated by reference inits entirety. This application is related to U.S. patent applicationSer. No. 13/853,001, titled “ENGINEERED LEATHER AND METHODS OFMANUFACTURE THEREOF” and filed on Mar. 28, 2013; U.S. patent applicationSer. No. 14/967,173, titled “ENGINEERED LEATHER AND METHODS OFMANUFACTURE THEREOF” and filed on Dec. 11, 2015; and PCT PatentApplication No. PCT/US2015/058794, titled “REINFORCED ENGINEEREDBIOMATERIALS AND METHODS OF MANUFACTURE THEREOF” and filed on Nov. 3,2015.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to biofabricated leather materials composed ofunbundled and randomly-oriented trimeric collagen fibrils that exhibitsuperior strength, non-anisotropic properties, and uniformity bycomparison to conventional leather products, but which have the look,feel and other aesthetic properties of natural leather. Unlike syntheticleather products composed of plastic resins, the biofabricated leatherof the invention is based on collagen, a natural component of leather.

Description of Related Art

Leather. Leather is used in a vast variety of applications, includingfurniture upholstery, clothing, shoes, luggage, handbag and accessories,and automotive applications. The estimated global trade value in leatheris approximately US $100 billion per year (Future Trends in the WorldLeather Products Industry and Trade, United Nations IndustrialDevelopment Organization, Vienna, 2010) and there is a continuing andincreasing demand for leather products. New ways to meet this demand arerequired in view of the economic, environmental and social costs ofproducing leather. To keep up with technological and aesthetic trends,producers and users of leather products seek new materials exhibitingsuperior strength, uniformity, processability and fashionable andappealing aesthetic properties that incorporate natural components.

Natural leathers are produced from the skins of animals which requireraising livestock. However, the raising of livestock requires enormousamounts of feed, pastureland, water, and fossil fuels. It also producesair and waterway pollution, including production of greenhouse gaseslike methane. Some states in the United States, such as California, mayimpose taxes on the amounts of pollutants such as methane produced bylivestock. As the costs of raising livestock rise, the cost of leatherwill rise.

The global leather industry slaughters more than a billion animals peryear. Most leather is produced in countries that engage in factoryfarming, lack animal welfare laws, or in which such laws go largely orcompletely unenforced. This slaughter under inhumane conditions isobjectionable to many socially conscious people. Consequently, there isa demand from consumers with ethical, moral or religious objections tothe use of natural leather products for products humanely producedwithout the mistreatment or slaughter of animals or produced in waysthat minimize the number of animals slaughtered.

The handling and processing of animal skins into leather also poseshealth risks because the handling animal skins can expose workers toanthrax and other pathogens and allergens such as those in leather dust.Factory farming of animals contributes to the spread of influenza (e.g.“bird flu”) and other infectious diseases that may eventually mutate andinfect humans. Animal derived products are also susceptible tocontamination with viruses and prions (“mad cow disease”). For producerand consumer peace of mind, there exists a demand for leather productsthat do not present these risks.

Natural leather is generally a durable and flexible material created byprocessing rawhide and skin of an animal, such as cattle hides. Thisprocessing typically involves three main parts: preparatory stages,tanning, and retanning. Leather may also be surface coated or embossed.

Numerous ways are known to prepare a skin or hide and convert it toleather. These include salting or refrigerating a hide or skin topreserve it; soaking or rehydrating the hide in an aqueous solution thatcontains surfactants or other chemicals to remove salt, dirt, debris,blood, and excess fat; defleshing or removing subcutaneous material fromthe hide; dehairing or unhairing the hide remove most of the hair;liming the hide to loosen fibers and open up collagen bundles allowingit to absorb chemicals; splitting the hide into two or more layers;deliming the hide to remove alkali and lower its pH; bating the hide tocomplete the deliming process and smooth the grain; degreasing to removeexcess fats; frizzing; bleaching; pickling by altering the pH; ordepickling,

Once the preparatory stages are complete, the leather is tanned. Leatheris tanned to increase its durability compared to untreated hide. Tanningconverts proteins in the hide or skin into a stable material that willnot putrefy while allowing the leather material to remain flexible.During tanning, the skin structure may be stabilized in an “open” formby reacting some of the collagen with complex ions of chromium or othertanning agents. Depending on the compounds used, the color and textureof the leather may change.

Tanning is generally understood to be the process of treating the skinsof animals to produce leather. Tanning may be performed in any number ofwell-understood ways, including by contacting a skin or hide with avegetable tanning agent, chromium compound, aldehyde, syntan, synthetic,semisynthetic or natural resin or polymer, or/and tanning natural oil ormodified oil. Vegetable tannins include pyrogallol- orpyrocatechin-based tannins, such as valonea, mimosa, ten, tara, oak,pinewood, sumach, quebracho and chestnut tannins; chromium tanningagents include chromium salts like chromium sulfate; aldehyde tanningagents include glutaraldehyde and oxazolidine compounds, syntans includearomatic polymers, polyacrylates, polymethacrylates, copolymers ofmaleic anhydride and styrene, condensation products of formaldehyde withmelamine or dicyandiamide, lignins and natural flours.

Chromium is the most commonly used tanning material. The pH of theskin/hide may be adjusted (e.g., lowered, e.g. to pH 2.8-3.2) to allowpenetration of the tanning agent; following penetration the pH may beraised to fix the tanning agent (“basification” to a slightly higherlevel, e.g., pH 3.8-4.2 for chrome).

After tanning, a leather may be retanned. Retanning refers to thepost-tanning treatment that can include coloring (dying), thinning,drying or hydrating, and the like. Examples of retanning techniquesinclude: tanning, wetting (rehydrating), sammying (drying),neutralization (adjusting pH to a less acidic or alkaline state),dyeing, fat liquoring, fixation of unbound chemicals, setting,conditioning, softening, buffing, etc.

A tanned leather product may be mechanically or chemically finished.Mechanical finishing can polish the leather to yield a shiny surface,iron and plate a leather to have a flat, smooth surface, emboss aleather to provide a three dimensional print or pattern, or tumble aleather to provide a more evident grain and smooth surface. Chemicalfinishing may involve the application of a film, a natural or syntheticcoating, or other leather treatment. These may be applied, for example,by spraying, curtain-coating or roller coating.

In animal hide, variations in fibrous collagen organization are observedin animals of different ages or species. These differences affect thephysical properties of hides and differences in leather produced fromthe hides. Variations in collagen organization also occur through thethickness of the hide. The top grain side of hide is composed of a finenetwork of collagen fibrils while deeper sections (corium) are composedof larger fiber bundles (FIG. 2). The smaller fibril organization of thegrain layer gives rise to a soft and smooth leather aesthetic while thelarger fiber bundle organization of deeper regions gives rise to a roughand course leather aesthetic.

The porous, fibrous organization of collagen in a hide allows appliedmolecules to penetrate, stabilize, and lubricate it during leathertanning. The combination of the innate collagen organization in hide andthe modifications achieved through tanning give rise to the desirablestrength, drape and aesthetic properties of leather.

The top grain surface of leather is often regarded as the most desirabledue to its smoothness and soft texture. This leather grain contains ahighly porous network of organized collagen fibrils. Endogenous collagenfibrils are organized to have lacunar regions and overlapping regions;see the hierarchical organization of collagen depicted by FIG. 1. Thestrengths, microscale porosity, and density of fibrils in a top grainleather allow tanning or fatliquoring agents to penetrate it, thusstabilizing and lubricating the collagen fibrils, producing a soft,smooth and strong leather that people desire.

Leather hides can be split to obtain leather that is mostly top grain.The split hide can be further abraded to reduce the coarser grainedcorium on the split side, but there is always some residual corium andassociated rough appearance. In order to produce leather with smoothgrain on both sides, it is necessary to combine two pieces of grain,corium side facing corium side and either sew them together or laminatethem with adhesives with the smooth top grain sides facing outward.There is a demand for a leather product that has a smooth, topgrain-like surface on both its sides, because this would avoid the needfor splitting, and sewing or laminating two split leather piecestogether.

Control of the final properties of leather is limited by the naturalvariation in collagen structure between different animal hides. Forexample, the relative thickness of grain to corium in goat hide issignificantly higher than that in kangaroo hide. In addition, the weaveangle of collagen fiber bundles in kangaroo corium are much moreparallel to the surface of the hide, while fiber bundles in bovinecorium are oriented in both parallel and perpendicular orientations tothe surface of the hide. Further, the density of fiber bundles varieswithin each hide depending on their anatomical location . Hide takenfrom butt, belly, shoulder, and neck can have different compositions andproperties. The age of an animal also affects the composition of itshide, for example, juvenile bovine hide contains smaller diameter fibersthan the larger fiber bundles found in adult bovine hide.

The final properties of leather can be controlled to some extent throughthe incorporation of stabilizing and lubricating molecules into the hideor skin during tanning and retanning, however, the selection of thesemolecules is limited by the need to penetrate the dense structure of theskin or hide. Particles as large as several microns in diameter havebeen incorporated into leather for enhanced lubrication; however,application of these particles is limited to hides with the largest poresizes. Uniformly distributing the particles throughout the hide presentsmany challenges.

Collagen. Collagen is a component of leather. Skin, or animal hide,contains significant amounts of collagen, a fibrous protein. Collagen isa generic term for a family of at least 28 distinct collagen types;animal skin is typically Type I collagen, although other types ofcollagen can be used in forming leather including type III collagen.

Collagens are characterized by a repeating triplet of amino acids,-(Gly-X-Y)_(n)- and approximately one-third of the amino acid residuesin collagen are glycine. X is often proline and Y is oftenhydroxyproline, though there may be up to 400 possible Gly-X-Y triplets.Different animals may produce different amino acid compositions of thecollagen, which may result in different properties and in differences inthe resulting leather.

The structure of collagen can consist of three intertwined peptidechains of differing lengths. Collagen triple helices (or monomers) maybe produced from alpha-chains of about 1,050 amino acids long, so thatthe triple helix takes the form of a rod of about approximately 300 nmlong, with a diameter of approximately 1.5 nm.

In the production of extracellular matrix by fibroblast skin cells,triple helix monomers may be synthesized and the monomers mayself-assemble into a fibrous form. These triple helices are heldtogether by electrostatic interactions including salt bridging, hydrogenbonding, Van der Waals interactions, dipole-dipole forces, polarizationforces, hydrophobic interactions, and/or covalent bonding. Triplehelices can be bound together in bundles called fibrils, and fibrils canfurther assemble to create fibers and fiber bundles (FIG. 1).

Fibrils have a characteristic banded appearance due to the staggeredoverlap of collagen monomers. The distance between bands isapproximately 67 nm for Type I collagen. Fibrils and fibers typicallybranch and interact with each other throughout a layer of skin.Variations of the organization or crosslinking of fibrils and fibers mayprovide strength to the material.

Fibers may have a range of diameters depending on the type of animalhide. In addition to type I collagen, skin (hides) may include othertypes of collagen as well, including type III collagen (reticulin), typeIV collagen, and type VII collagen.

Various types of collagen exist throughout the mammalian body. Forexample, besides being the main component of skin and animal hide, TypeI collagen also exists in cartilage, tendon, vascular ligature, organs,muscle, and the organic portion of bone. Successful efforts have beenmade to isolate collagen from various regions of the mammalian body inaddition to the animal skin or hide. Decades ago, researchers found thatat neutral pH, acid-solubilized collagen self-assembled into fibrilscomposed of the same cross-striated patterns observed in native tissue;Schmitt F. O. J. Cell. Comp Physiol. 1942; 20:11). This led to use ofcollagen in tissue engineering and a variety of biomedical applications.In more recent years, collagen has been harvested from bacteria andyeast using recombinant techniques.

Regardless of the type of collagen, all are formed and stabilizedthrough a combination of physical and chemical interactions includingelectrostatic interactions including salt bridging, hydrogen bonding,Van der Waals interactions, dipole-dipole forces, polarization forces,hydrophobic interactions, and covalent bonding often catalyzed byenzymatic reactions. For Type I collagen fibrils, fibers, and fiberbundles, its complex assembly is achieved in vivo during development andis critical in providing mechanical support to the tissue while allowingfor cellular motility and nutrient transport. Various distinct collagentypes have been identified in vertebrates. These include bovine, ovine,porcine, chicken, and human collagens.

Generally, the collagen types are numbered by Roman numerals, and thechains found in each collagen type are identified by Arabic numerals.Detailed descriptions of structure and biological functions of thevarious different types of naturally occurring collagens are availablein the art; see, e.g., Ayad et al. (1998) The Extracellular Matrix FactsBook, Academic Press, San Diego, Calif.; Burgeson, R. E., and Nimmi(1992) “Collagen types: Molecular Structure and Tissue Distribution” inClin. Orthop. 282:250-272; Kielty, C. M. et al. (1993) “The CollagenFamily: Structure, Assembly And Organization In The ExtracellularMatrix,” Connective Tissue And Its Heritable Disorders, MolecularGenetics, And Medical Aspects, Royce, P. M. and B. Steinmann eds.,Wiley-Liss, N.Y., pp. 103-147; and Prockop, D. J- and K. I. Kivirikko(1995) “Collagens: Molecular Biology, Diseases, and Potentials forTherapy,” Annu. Rev. Biochem., 64:403-434.)

Type I collagen is the major fibrillar collagen of bone and skincomprising approximately 80-90% of an organism's total collagen. Type Icollagen is the major structural macromolecule present in theextracellular matrix of multicellular organisms and comprisesapproximately 20% of total protein mass. Type I collagen is aheterotrimeric molecule comprising two α1(I) chains and one α2(I) chain,encoded by the COL1A1 and COL1A2 genes, respectively. Other collagentypes are less abundant than type I collagen, and exhibit differentdistribution patterns. For example, type II collagen is the predominantcollagen in cartilage and vitreous humor, while type III collagen isfound at high levels in blood vessels and to a lesser extent in skin.

Type II collagen is a homotrimeric collagen comprising three identicala1(II) chains encoded by the COL2A1 gene. Purified type II collagen maybe prepared from tissues by, methods known in the art, for example, byprocedures described in Miller and Rhodes (1982) Methods In Enzymology82:33-64.

Type III collagen is a major fibrillar collagen found in skin andvascular tissues. Type III collagen is a homotrimeric collagencomprising three identical a1(III) chains encoded by the COL3A1 gene.Methods for purifying type III collagen from tissues can be found in,for example, Byers et al. (1974) Biochemistry 13:5243-5248; and Millerand Rhodes, supra.

Type IV collagen is found in basement membranes in the form of sheetsrather than fibrils. Most commonly, type IV collagen contains two α1(IV)chains and one α2(IV) chain. The particular chains comprising type IVcollagen are tissue-specific. Type IV collagen may be purified using,for example, the procedures described in Furuto and Miller (1987)Methods in Enzymology, 144:41-61, Academic Press.

Type V collagen is a fibrillar collagen found in, primarily, bones,tendon, cornea, skin, and blood vessels. Type V collagen exists in bothhomotrimeric and heterotrimeric forms. One form of type V collagen is aheterotrimer of two α1(V) chains and one α2(V) chain. Another form oftype V collagen is a heterotrimer of α1(V), α2(V), and α3(V) chains. Afurther form of type V collagen is a homotrimer of α1(V). Methods forisolating type V collagen from natural sources can be found, forexample, in Elstow and Weiss (1983) Collagen Rel. Res. 3:181-193, andAbedin et al. (1982) Biosci. Rep. 2:493-502.

Type VI collagen has a small triple helical region and two largenon-collagenous remainder portions. Type VI collagen is a heterotrimercomprising α1(VI), α2(VI), and α3(VI) chains. Type VI collagen is foundin many connective tissues. Descriptions of how to purify type VIcollagen from natural sources can be found, for example, in Wu et al.(1987) Biochem. J. 248:373-381, and Kielty et al. (1991) J. Cell Sci.99:797-807.

Type VII collagen is a fibrillar collagen found in particular epithelialtissues. Type VII collagen is a homotrimeric molecule of three al(VII)chains. Descriptions of how to purify type VII collagen from tissue canbe found in, for example, Lunstrum et al. (1986) J. Biol. Chem.261:9042-9048, and Bentz et al. (1983) Proc. Natl. Acad. Sci. USA80:3168-3172.Type VIII collagen can be found in Descemet's membrane inthe cornea. Type VIII collagen is a heterotrimer comprising two α1(VIII)chains and one α2(VIII) chain, although other chain compositions havebeen reported. Methods for the purification of type VIII collagen fromnature can be found, for example, in Benya and Padilla (1986) J. Biol.Chem. 261:4160-4169, and Kapoor et al. (1986) Biochemistry 25:3930-3937.

Type IX collagen is a fibril-associated collagen found in cartilage andvitreous humor. Type IX collagen is a heterotrimeric molecule comprisingα1(IX), α2(IX), and α3 (IX) chains. Type IX collagen has been classifiedas a FACIT (Fibril Associated Collagens with Interrupted Triple Helices)collagen, possessing several triple helical domains separated bynon-triple helical domains. Procedures for purifying type IX collagencan be found, for example, in Duance, et al. (1984) Biochem. J.221:885-889; Ayad et al. (1989) Biochem. J. 262:753-761; and Grant etal. (1988) The Control of Tissue Damage, Glauert, A. M., ed., ElsevierScience Publishers, Amsterdam, pp. 3-28.

Type X collagen is a homotrimeric compound of α1(X) chains. Type Xcollagen has been isolated from, for example, hypertrophic cartilagefound in growth plates; see, e.g., Apte et al. (1992) Eur J Biochem 206(1):217-24.

Type XI collagen can be found in cartilaginous tissues associated withtype II and type IX collagens, and in other locations in the body. TypeXI collagen is a heterotrimeric molecule comprising α1(XI), α2(XI), andα3(XI) chains. Methods for purifying type XI collagen can be found, forexample, in Grant et al., supra.

Type XII collagen is a FACIT collagen found primarily in associationwith type I collagen. Type XII collagen is a homotrimeric moleculecomprising three α1(XII) chains. Methods for purifying type XII collagenand variants thereof can be found, for example, in Dublet et al. (1989)J. Biol. Chem. 264:13150-13156; Lunstrum et al. (1992) J. Biol. Chem.267:20087-20092; and Watt et al. (1992) J. Biol. Chem. 267:20093-20099.

Type XIII is a non-fibrillar collagen found, for example, in skin,intestine, bone, cartilage, and striated muscle. A detailed descriptionof type XIII collagen may be found, for example, in Juvonen et al.(1992) J. Biol. Chem. 267: 24700-24707.

Type XIV is a FACIT collagen characterized as a homotrimeric moleculecomprising α1(XIV) chains. Methods for isolating type XIV collagen canbe found, for example, in Aubert-Foucher et al. (1992) J. Biol. Chem.267:15759-15764,and Watt et al., supra.

Type XV collagen is homologous in structure to type XVIII collagen.Information about the structure and isolation of natural type XVcollagen can be found, for example, in Myers et al. (1992) Proc. Natl.Acad. Sci. USA 89:10144-10148; Huebner et al. (1992) Genomics14:220-224; Kivirikko et al. (1994) J. Biol. Chem. 269:4773-4779; andMuragaki, J. (1994) Biol. Chem. 264:4042-4046.

Type XVI collagen is a fibril-associated collagen, found, for example,in skin, lung fibroblast, and keratinocytes. Information on thestructure of type XVI collagen and the gene encoding type XVI collagencan be found, for example, in Pan et al. (1992) Proc. Natl. Acad. Sci.USA 89:6565-6569; and Yamaguchi et al. (1992) J. Biochem. 112:856-863.

Type XVII collagen is a hemidesmosal transmembrane collagen, also knownat the bullous pemphigoid antigen. Information on the structure of typeXVII collagen and the gene encoding type XVII collagen can be found, forexample, in Li et al. (1993) J. Biol. Chem. 268(12):8825-8834; andMcGrath et al. (1995) Nat. Genet. 11(1):83-86.

Type XVIII collagen is similar in structure to type XV collagen and canbe isolated from the liver. Descriptions of the structures and isolationof type XVIII collagen from natural sources can be found, for example,in Rehn and Pihlajaniemi (1994) Proc. Natl. Acad. Sci USA 91:4234-4238;Oh et al. (1994) Proc. Natl. Acad. Sci USA 91:4229-4233; Rehn et al.(1994) J. Biol. Chem. 269:13924-13935; and Oh et al. (1994) Genomics19:494-499.

Type XIX collagen is believed to be another member of the FACIT collagenfamily, and has been found in mRNA isolated from rhabdomyosarcoma cells.Descriptions of the structures and isolation of type XIX collagen can befound, for example, in Inoguchi et al. (1995) J. Biochem. 117:137-146;Yoshioka et al. (1992) Genomics 13:884-886; and Myers et al., J. Biol.Chem. 289:18549-18557 (1994).

Type XX collagen is a newly found member of the FACIT collagenousfamily, and has been identified in chick cornea. (See, e.g., Gordon etal. (1999) FASEB Journal 13:A1119; and Gordon et al. (1998), IOVS39:S1128.)

Any type of collagen, truncated collagen, unmodified orpost-translationally modified, or amino acid sequence-modified collagenthat can be fibrillated and crosslinked by the methods described hereincan be used to produce a biofabricated material or biofabricatedleather. Biofabricated leather may contain a substantially homogenouscollagen, such as only Type I or Type III collagen or may containmixtures of 2, 3, 4 or more different kinds of collagens.

Recombinant Collagen. Recombinant expression of collagen andcollagen-like proteins is known and is incorporated by reference toBell, EP 1232182B1, Bovine collagen and method for producing recombinantgelatin; Olsen, et al., U.S. Pat. No. 6,428,978, Methods for theproduction of gelatin and full-length triple helical collagen inrecombinant cells; VanHeerde, et al., U.S. Pat. No. 8,188,230, Methodfor recombinant microorganism expression and isolation of collagen-likepolypeptides. Such recombinant collagens have not been used to produceleather.

Prokaryotic expression. In prokaryotic systems, such as bacterialsystems, a number of expression vectors may be advantageously selecteddepending upon the use intended for the expressed polypeptide. Forexample, when large quantities of the animal collagens and gelatins ofthe invention are to be produced, such as for the generation ofantibodies, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include, but are not limited to, the E. coli expression vectorpUR278 (Ruther et al. (1983) EMBO J. 2:1791), in which the codingsequence may be ligated into the vector in frame with the lac Z codingregion so that a hybrid AS-lacZ protein is produced; pIN vectors (Inouyeet al. (1985) Nucleic Acids Res. 13:3101-3109 and Van Heeke et al.(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors mayalso be used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned polypeptide of interestcan be released from the GST moiety. A recombinant collagen may comprisecollagen molecules that have not been post-translationally modified,e.g., not glycosylated or hydroxylated, or may comprise one or morepost-translational modifications, for example, modifications thatfacilitate fibrillation and formation of unbundled and randomly orientedfibrils of collagen molecules.

A recombinant collagen molecule can comprise a fragment of the aminoacid sequence of a native collagen molecule that can form trimericcollagen fibrils or a modified collagen molecule or truncated collagenmolecule having an amino acid sequence at least 70, 80, 90, 95, 96, 97,98, or 99% identical or similar to a native collagen amino acid sequence(or to a fibril forming region thereof or to a segment substantiallycomprising [Gly-X-Y]_(n)) , such as those of bovine collagen, describedby SEQ ID NOS: 1, 2 or 3 and by amino acid sequences of Col1A1, Col1A2,and Col1A3, described by Accession Nos. NP 001029211.1(https://_www.ncbi.nlm.nih.gov/protein/77404252, last accessed Feb. 9,2017), NP_776945.1 (https://_www.ncbi.nlm.nih.gov/protein/27806257 lastaccessed Feb. 9, 2017) and NP_001070299.1(https://_www.ncbi.nlm.nih.gov/protein/116003881 last accessed Feb. 9,2017) which are incorporated by reference. (These links have beeninactivated by inclusion of an underline after the double slash.)

Such recombinant or modified collagen molecules will generally comprisethe repeated -(Gly-X-Y)_(n)- sequence described herein.

BLASTN may be used to identify a polynucleotide sequence having at least70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, or 99% sequenceidentity to a reference polynucleotide such as a polynucleotide encodinga collagen polypeptide or encoding the amino acid sequences of SEQ IDNOS: 1, 2 or 3. A representative BLASTN setting optimized to find highlysimilar sequences uses an Expect Threshold of 10 and a Wordsize of 28,max matches in query range of 0, match/mismatch scores of 1/−2, andlinear gap cost. Low complexity regions may be filtered or masked.Default settings of a Standard Nucleotide BLAST are described by andincorporated by reference tohttps://_blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome(last accessed Jan. 27, 2017).

BLASTP can be used to identify an amino acid sequence having at least70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, or 99% sequenceidentity, or similarity to a reference amino acid, such as a collagenamino acid sequence, using a similarity matrix such as BLOSUM45,BLOSUM62 or BLOSUM80 where BLOSUM45 can be used for closely relatedsequences, BLOSUM62 for midrange sequences, and BLOSUM80 for moredistantly related sequences. Unless otherwise indicated a similarityscore will be based on use of BLOSUM62. When BLASTP is used, the percentsimilarity is based on the BLASTP positives score and the percentsequence identity is based on the BLASTP identities score. BLASTP“Identities” shows the number and fraction of total residues in the highscoring sequence pairs which are identical; and BLASTP “Positives” showsthe number and fraction of residues for which the alignment scores havepositive values and which are similar to each other. Amino acidsequences having these degrees of identity or similarity or anyintermediate degree of identity or similarity to the amino acidsequences disclosed herein are contemplated and encompassed by thisdisclosure. A representative BLASTP setting that uses an ExpectThreshold of 10, a Word Size of 3, BLOSUM 62 as a matrix, and GapPenalty of 11 (Existence) and 1 (Extension) and a conditionalcompositional score matrix adjustment. Other default settings for BLASTPare described by and incorporated by reference to the disclosureavailable at:https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINKLOC=blasthome (last accessed Jan. 27, 2017).

Yeast expression. In one embodiment, collagen molecules are produced ina yeast expression system. In yeast, a number of vectors containingconstitutive or inducible promoters known in the art may be used;Ausubel et al., supra, Vol. 2, Chapter 13; Grant et al. (1987)Expression and Secretion Vectors for Yeast, in Methods in Enzymology,Ed. Wu & Grossman, Acad. Press, N.Y. 153:516-544; Glover (1986) DNACloning, Vol. II, IRL Press, Wash., D.C., Ch. 3; Bitter (1987)Heterologous Gene Expression in Yeast, in Methods in Enzymology, Eds.Berger & Kimmel, Acad. Press, N.Y. 152:673-684; and The MolecularBiology of the Yeast Saccharomyces, Eds. Strathern et al., Cold SpringHarbor Press, Vols. I and II (1982).

Collagen can be expressed using host cells, for example, from the yeastSaccharomyces cerevisiae. This particular yeast can be used with any ofa large number of expression vectors. Commonly employed expressionvectors are shuttle vectors containing the 2P origin of replication forpropagation in yeast and the Col E1 origin for E. coli, for efficienttranscription of the foreign gene. A typical example of such vectorsbased on 2P plasmids is pWYG4, which has the 2P ORI-STB elements, theGAL1-10 promoter, and the 2P D gene terminator. In this vector, an Ncolcloning site is used to insert the gene for the polypeptide to beexpressed, and to provide the ATG start codon. Another expression vectoris pWYG7L, which has intact 2αORI, STB, REP1 and REP2, and the GAL1-10promoter, and uses the FLP terminator. In this vector, the encodingpolynucleotide is inserted in the polylinker with its 5′ ends at a BamHIor Ncol site. The vector containing the inserted polynucleotide istransformed into S. cerevisiae either after removal of the cell wall toproduce spheroplasts that take up DNA on treatment with calcium andpolyethylene glycol or by treatment of intact cells with lithium ions.

Alternatively, DNA can be introduced by electroporation. Transformantscan be selected, for example, using host yeast cells that areauxotrophic for leucine, tryptophan, uracil, or histidine together withselectable marker genes such as LEU2, TRP1, URA3, HIS3, or LEU2-D.

In one embodiment, polynucleotides encoding collagen are introduced intohost cells from the yeast Pichia. Species of non-Saccharomyces yeastsuch as Pichia pastoris appear to have special advantages in producinghigh yields of recombinant protein in scaled up procedures.Additionally, a Pichia expression kit is available from InvitrogenCorporation (San Diego, Calif.).

There are a number of methanol responsive genes in methylotrophic yeastssuch as Pichia pastoris, the expression of each being controlled bymethanol responsive regulatory regions, also referred to as promoters.Any of such methanol responsive promoters are suitable for use in thepractice of the present invention. Examples of specific regulatoryregions include the AOX1 promoter, the AOX2 promoter, thedihydroxyacetone synthase (DAS), the P40 promoter, and the promoter forthe catalase gene from P. pastoris, etc.

In other embodiments, the methylotrophic yeast Hansenula polymorpha isused. Growth on methanol results in the induction of key enzymes of themethanol metabolism, such as MOX (methanol oxidase), DAS(dihydroxyacetone synthase), and FMHD (formate dehydrogenase). Theseenzymes can constitute up to 30-40% of the total cell protein. The genesencoding MOX, DAS, and FMDH production are controlled by strongpromoters induced by growth on methanol and repressed by growth onglucose. Any or all three of these promoters may be used to obtainhigh-level expression of heterologous genes in H. polymorpha. Therefore,in one aspect, a polynucleotide encoding animal collagen or fragments orvariants thereof is cloned into an expression vector under the controlof an inducible H. polymorpha promoter. If secretion of the product isdesired, a polynucleotide encoding a signal sequence for secretion inyeast is fused in frame with the polynucleotide. In a furtherembodiment, the expression vector preferably contains an auxotrophicmarker gene, such as URA3 or LEU2, which may be used to complement thedeficiency of an auxotrophic host.

The expression vector is then used to transform H. polymorpha host cellsusing techniques known to those of skill in the art. A useful feature ofH. polymorpha transformation is the spontaneous integration of up to 100copies of the expression vector into the genome. In most cases, theintegrated polynucleotide forms multimers exhibiting a head-to-tailarrangement. The integrated foreign polynucleotide has been shown to bemitotically stable in several recombinant strains, even undernon-selective conditions. This phenomena of high copy integrationfurther ads to the high productivity potential of the system.

Fungal Expression. Filamentous fungi may also be used to produce thepresent polypeptides. Vectors for expressing and/or secretingrecombinant proteins in filamentous fungi are well known, and one ofskill in the art could use these vectors to express the recombinantanimal collagens of the present invention.

Plant Expression. In one aspect, an animal collagen is produced in aplant or plant cells. In cases where plant expression vectors are used,the expression of sequences encoding the collagens of the invention maybe driven by any of a number of promoters. For example, viral promoterssuch as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al. (1984)Nature 310:511-514), or the coat protein promoter of TMV (Takamatsu etal. (1987) EMBO J. 6:307-311) may be used; alternatively, plantpromoters such as the small subunit of RUBISCO (Coruzzi et al. (1984)EMBO J. 3:1671-1680; Broglie et al. (1984) Science 224:838-843) or heatshock promoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al.(1986) Mol. Cell. Biol. 6:559-565) may be used. These constructs can beintroduced into plant cells by a variety of methods known to those ofskill in the art, such as by using Ti plasmids, Ri plasmids, plant virusvectors, direct DNA transformation, microinjection, electroporation,etc. For reviews of such techniques see, for example, Weissbach &Weissbach, Methods for Plant Molecular Biology, Academic Press, NY,Section VIII, pp. 421-463 (1988); Grierson & Corey, Plant MolecularBiology, 2d Ed., Blackie, London, Ch. 7-9 (1988); Transgenic Plants: AProduction System for Industrial and Pharmaceutical Proteins, Owen andPen eds., John Wiliey & Sons, 1996; Transgenic Plants, Galun and Breimaneds, Imperial College Press, 1997; and Applied Plant Biotechnology,Chopra, Malik, and Bhat eds., Science Publishers, Inc., 1999.

Plant cells do not naturally produce sufficient amounts ofpost-translational enzymes to efficiently produce stable collagen.Therefore, where hydroxylation is desired, plant cells used to expressanimal collagens are supplemented with the necessary post-translationalenzymes to sufficiently produce stable collagen. In a preferredembodiment of the present invention, the post-translational enzyme isprolyl 4-hydroxylase.

Methods of producing the present animal collagens in plant systems maybe achieved by providing a biomass from plants or plant cells, whereinthe plants or plant cells comprise at least one coding sequence isoperably linked to a promoter to effect the expression of thepolypeptide, and the polypeptide is then extracted from the biomass.Alternatively, the polypeptide can be non-extracted, e.g., expressedinto the endosperm.

Plant expression vectors and reporter genes are generally known in theart; see, e.g., Gruber et al. (1993) in Methods of Plant MolecularBiology and Biotechnology, CRC Press. Typically, the expression vectorcomprises a nucleic acid construct generated, for example, recombinantlyor synthetically, and comprising a promoter that functions in a plantcell, wherein such promoter is operably linked to a nucleic acidsequence encoding an animal collagen or fragments or variants thereof,or a post-translational enzyme important to the biosynthesis ofcollagen.

Promoters drive the level of protein expression in plants. To produce adesired level of protein expression in plants, expression may be underthe direction of a plant promoter. Promoters suitable for use inaccordance with the present invention are generally available in theart; see, e.g., PCT Publication No. WO 91/19806. Examples of promotersthat may be used in accordance with the present invention includenon-constitutive promoters or constitutive promoters. These promotersinclude, but are not limited to, the promoter for the small subunit ofribulose-1,5-bis-phosphate carboxylase; promoters from tumor-inducingplasmids of Agrobacterium tumefaciens, such as the RUBISCO nopalinesynthase (NOS) and octopine synthase promoters; bacterial T-DNApromoters such as mas and ocs promoters; and viral promoters such as thecauliflower mosaic virus (CaMV) 19S and 35S promoters or the figwortmosaic virus 35S promoter.

The polynucleotide sequences of the present invention can be placedunder the transcriptional control of a constitutive promoter, directingexpression of the collagen or post-translational enzyme in most tissuesof a plant. In one embodiment, the polynucleotide sequence is under thecontrol of the cauliflower mosaic virus (CaMV) 35S promoter. The doublestranded caulimorvirus family has provided the single most importantpromoter expression for transgene expression in plants, in particular,the 35S promoter; see, e.g., Kay et al. (1987) Science 236:1299.Additional promoters from this family such as the figwort mosaic viruspromoter, etc., have been described in the art, and may also be used;see, e.g., Sanger et al. (1990) Plant Mol. Biol. 14:433-443; Medberry etal. (1992) Plant Cell 4:195-192; and Yin and Beachy (1995) Plant J.7:969-980.

The promoters used in polynucleotide constructs for expressing collagenmay be modified, if desired, to affect their control characteristics.For example, the CaMV promoter may be ligated to the portion of theRUBISCO gene that represses the expression of RUBISCO in the absence oflight, to create a promoter which is active in leaves, but not in roots.The resulting chimeric promoter may be used as described herein.

Constitutive plant promoters having general expression properties knownin the art may be used with the expression vectors of the presentinvention. These promoters are abundantly expressed in most planttissues and include, for example, the actin promoter and the ubiquitinpromoter; see, e.g., McElroy et al. (1990) Plant Cell 2:163-171; andChristensen et al. (1992) Plant Mol. Biol. 18:675-689.

Alternatively, the polypeptide of the present invention may be expressedin a specific tissue, cell type, or under more precise environmentalconditions or developmental control. Promoters directing expression inthese instances are known as inducible promoters. In the case where atissue-specific promoter is used, protein expression is particularlyhigh in the tissue from which extraction of the protein is desired.Depending on the desired tissue, expression may be targeted to theendosperm, aleurone layer, embryo (or its parts as scutellum andcotyledons), pericarp, stem, leaves tubers, roots, etc. Examples ofknown tissue-specific promoters include the tuber-directed class Ipatatin promoter, the promoters associated with potato tuber ADPGPPgenes, the soybean promoter of β-conglycinin (7S protein) which drivesseed-directed transcription, and seed-directed promoters from the zeingenes of maize endosperm; see, e.g., Bevan et al. (1986) Nucleic AcidsRes. 14: 4625-38; Muller et al. (1990) Mol. Gen. Genet. 224:136-46; Bray(1987) Planta 172: 364-370; and Pedersen et al. (1982) Cell 29:1015-26.

Collagen polypeptides can be produced in seed by way of seed-basedproduction techniques using, for example, canola, corn, soybeans, riceand barley seed. In such a process, for example, the product isrecovered during seed germination; see, e.g., PCT Publication Numbers WO9940210; WO 9916890; WO 9907206;U.S. Pat. No. 5,866,121; U.S. Pat. No.5,792,933; and all references cited therein. Promoters that may be usedto direct the expression of the polypeptides may be heterologous ornon-heterologous. These promoters can also be used to drive expressionof antisense nucleic acids to reduce, increase, or alter concentrationand composition of the present animal collagens in a desired tissue.

Other modifications that may be made to increase and/or maximizetranscription of the present polypeptides in a plant or plant cell arestandard and known to those in the art. For example a vector comprisinga polynucleotide sequence encoding a recombinant animal collagen, or afragment or variant thereof, operably linked to a promoter may furthercomprise at least one factor that modifies the transcription rate ofcollagen or related post-translational enzymes, including, but notlimited to, peptide export signal sequence, codon usage, introns,polyadenylation, and transcription termination sites. Methods ofmodifying constructs to increase expression levels in plants aregenerally known in the art; see, e.g. Rogers et al. (1985) J. Biol.Chem. 260:3731; and Cornejo et al. (1993) Plant Mol Biol 23:567-58. Inengineering a plant system that affects the rate of transcription of thepresent collagens and related post-translational enzymes, variousfactors known in the art, including regulatory sequences such aspositively or negatively acting sequences, enhancers and silencers, aswell as chromatin structure can affect the rate of transcription inplants. At least one of these factors may be utilized when expressing arecombinant animal collagen, including but not limited to the collagentypes described above.

The vectors comprising the present polynucleotides will typicallycomprise a marker gene which confers a selectable phenotype on plantcells. Usually, the selectable marker gene will encode antibioticresistance, with suitable genes including at least one set of genescoding for resistance to the antibiotic spectinomycin, the streptomycinphophotransferase (SPT) gene coding for streptomycin resistance, theneomycin phophotransferase (NPTH) gene encoding kanamycin or geneticinresistance, the hygromycin resistance, genes coding for resistance toherbicides which act to inhibit the action of acetolactate synthase(ALS), in particular, the sulfonylurea-type herbicides; e.g., theacetolactate synthase (ALS) gene containing mutations leading to suchresistance in particular the S4 and/or Hra mutations, genes coding forresistance to herbicides which act to inhibit action of glutaminesynthase, such as phophinothricin or basta; e.g. the bar gene, or othersimilar genes known in the art. The bar gene encodes resistance to theherbicide basta, the nptll gene encodes resistance to the antibioticskanamycin and geneticin, and the ALS gene encodes resistance to theherbicide chlorsulfuron.

Typical vectors useful for expression of foreign genes in plants arewell known in the art, including, but not limited to, vectors derivedfrom the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. Thesevectors are plant integrating vectors that upon transformation,integrate a portion of the DNA into the genome of the host plant; seee.g., Rogers et al. (1987) Meth In Enzymol. 153:253-277; Schardl et al.(1987) Gene 61:1-11; and Berger et al., Proc. Natl. Acad. Sci. U.S.A.86:8402-8406.

Vectors comprising sequences encoding the present polypeptides andvectors comprising post-translational enzymes or subunits thereof may beco-introduced into the desired plant. Procedures for transforming plantcells are available in the art, for example, direct gene transfer, invitro protoplast transformation, plant virus-mediated transformation,liposome-mediated transformation, microinjection, electroporation,Agrobacterium mediated transformation, and particle bombardment; seee.g., Paszkowski et al. (1984) EMBO J. 3:2717-2722; U.S. Pat. No.4,684,611; European Application No. 0 67 553; U.S. Pat. No. 4,407,956;U.S. Pat. No. 4,536,475; Crossway et al. (1986) Biotechniques 4:320-334;Riggs et al. (1986) Proc. Natl. Acad. Sci USA 83:5602-5606; Hinchee etal. (1988) Biotechnology 6:915-921; and U.S. Pat. No. 4,945,050.)Standard methods for the transformation of, e.g., rice, wheat, corn,sorghum, and barley are described in the art; see, e.g., Christou et al.(1992) Trends in Biotechnology 10: 239 and Lee et al. (1991) Proc. Nat'lAcad. Sci. USA 88:6389. Wheat can be transformed by techniques similarto those employed for transforming corn or rice. Furthermore, Casas etal. (1993) Proc. Nat'l Acad. Sci. USA 90:11212, describe a method fortransforming sorghum, while Wan et al. (1994) Plant Physiol. 104: 37,teach a method for transforming barley. Suitable methods for corntransformation are provided by Fromm et al. (1990) Bio/Technology 8:833and by Gordon-Kamm et al., supra.

Additional methods that may be used to generate plants that produceanimal collagens of the present invention are established in the art;see, e.g., U.S. Pat. No. 5,959,091; U.S. Pat. No. 5,859,347; U.S. Pat.No.5,763,241; U.S. Pat. No. 5,659,122; U.S. Pat. No. 5,593,874; U.S.Pat. No. 5,495,071; U.S. Pat. No. 5,424,412; U.S. Pat. No. 5,362,865;U.S. Pat. No. 5,229,112; U.S. Pat. No. 5,981,841; U.S. Pat. No.5,959,179; U.S. Pat. No. 5,932,439; U.S. Pat. No. 5,869,720; U.S. Pat.No. 5,804,425; U.S. Pat. No. 5,763,245; U.S. Pat. No. 5,716,837; U.S.Pat. No. 5,689,052; U.S. Pat. No. 5,633,435; U.S. Pat. No. 5,631,152;U.S. Pat. No.5,627,061; U.S. Pat. No. 5,602,321; U.S. Pat. No.5,589,612; U.S. Pat. No. 5,510,253; U.S. Pat. No. 5,503,999; U.S. Pat.No. 5,378,619; U.S. Pat. No. 5,349,124; U.S. Pat. No. 5,304,730; U.S.Pat. No. 5,185,253; U.S. Pat. No. 4,970,168; European Publication No.EPA 00709462; European Publication No. EPA 00578627; EuropeanPublication No. EPA 00531273; European Publication No. EPA 00426641; PCTPublication No. WO 99/31248; PCT Publication No. WO 98/58069; PCTPublication No. WO 98/45457; PCT Publication No. WO 98/31812; PCTPublication No. WO 98/08962; PCT Publication No. WO 97/48814; PCTPublication No. WO 97/30582; and PCT Publication No. WO 9717459.

Insect Expression. Another alternative expression system for collagen isan insect system. Baculoviruses are very efficient expression vectorsfor the large scale production of various recombinant proteins in insectcells. The methods as described in Luckow et al. (1989) Virology170:31-39 and Gruenwald, S. and Heitz, J. (1993) Baculovirus ExpressionVector System: Procedures & Methods Manual, Pharmingen, San Diego,Calif., can be employed to construct expression vectors containing acollagen coding sequence for the collagens of the invention and theappropriate transcriptional/translational control signals. For example,recombinant production of proteins can be achieved in insect cells, byinfection of baculovirus vectors encoding the polypeptide. Theproduction of recombinant collagen , collagen-like or collagenouspolypeptides with stable triple helices can involve the co-infection ofinsect cells with three baculoviruses, one encoding the animal collagento be expressed and one each encoding the α subunit and β subunit ofprolyl 4-hydroxylase. This insect cell system allows for production ofrecombinant proteins in large quantities. In one such system, Autographacalifornica nuclear polyhidrosis virus (AcNPV) is used as a vector toexpress foreign genes. This virus grows in Spodoptera frugiperda cells.Coding sequences for collagen or collagen-like polypeptides may becloned into non-essential regions (for example the polyhedron gene) ofthe virus and placed under control of an AcNPV promoter (for example,the polyhedron promoter). Successful insertion of a coding sequence willresult in inactivation of the polyhedron gene and production ofnon-occluded recombinant virus; e.g., viruses lacking the proteinaceouscoat coded for by the polyhedron gene. These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed; see, e.g., Smith et al. (1983) J. Virol. 46:584; andU.S. Pat. No. 4,215,051. Further examples of this expression system maybe found in, for example, Ausubel et al. above.

Animal Expression. In animal host cells, a number of expression systemsmay be utilized. In cases where an adenovirus is used as an expressionvector, polynucleotide sequences encoding collagen or collagen-likepolypeptides may be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing the encoded polypeptidesin infected hosts; see, e.g., Logan & Shenk, Proc. Natl. Acad. Sci. USA81:3655-3659 (1984). Alternatively, the vaccinia 7.5 K promoter may beused; see, e.g., Mackett et al. (1982) Proc. Natl. Acad. Sci. USA79:7415-7419; Mackett et al. (1982) J. Virol. 49:857-864; and Panicaliet al. (1982) Proc. Natl. Acad. Sci. USA 79:4927-4931.

A preferred expression system in mammalian host cells is the SemlikiForest virus. Infection of mammalian host cells, for example, babyhamster kidney (BHK) cells and Chinese hamster ovary (CHO) cells canyield very high recombinant expression levels. Semliki Forest virus is apreferred expression system as the virus has a broad host range suchthat infection of mammalian cell lines will be possible. Morespecifically, Semliki Forest virus can be used in a wide range of hosts,as the system is not based on chromosomal integration, and thus providesan easier way of obtaining modifications of the recombinant animalcollagens in studies aiming at identifying structure functionrelationships and testing the effects of various hybrid molecules.Methods for constructing Semliki Forest virus vectors for expression ofexogenous proteins in mammalian host cells are described in, forexample, Olkkonen et al. (1994) Methods Cell Biol 43:43-53.

Non-human transgenic animals may also be used to express thepolypeptides of the present invention. Such systems can be constructedby operably linking the polynucleotide of the invention to a promoter,along with other required or optional regulatory sequences capable ofeffecting expression in mammary glands. Likewise, required or optionalpost-translational enzymes may be produced simultaneously in the targetcells employing suitable expression systems. Methods of using non-humantransgenic animals to recombinantly produce proteins are known in theart; see, e.g., U.S. Pat. No. 4,736,866; U.S. Pat. No. 5,824,838; U.S.Pat. No. 5,487,992; and U.S. Pat. No. 5,614,396.

The references cited in the sections above which describe the productionof recombinant collagens are each incorporated by reference.

Composite collagen fiber sheets. As shown in FIG. 1, triple helicalcollagen molecules associate into fibrils which in animal skin assembleinto larger fibril bundles or collagen fibers. Prior methods of makingcollagen sheets used a mixture of ground animal skin or leather scrapsand dissolved or suspended collagen. Such collagen fiber-containingproducts are described by U.S. Pat. Nos. 2,934,446; 3,073,714; 3,122,599; and 3,136,682. Highberger, et al., U.S. Pat. No. 2,934,446describes a method using a meat grinder to produce a slurry of calfskinhide or corium which is formed into a sheet, tanned and for forminginterlocked collagen fiber masses by comminuting and dispersing animalskin in an acidic aqueous solution at 5° C. and then raising the pH andtemperature to precipitate collagen fibers to form a gel which is thendried. These sheets of collagen fiber masses make use of leather scrapsand form sheets resembling leather. Highberger does not show that theseleather sheets are suitable for commercial use. Tu, et al., U.S. Pat.No. 3,073,714 discloses producing a sheet from an calfskin slurrycontaining 25% solids which is tanned with a vegetable tanning solutionand treated with glycerin and oleic acid. These collagen fiber sheetsare described as reproducing the internal arrangement of collagen fibersin natural skins and hides. Tu does not show that the leather sheets arecompositionally or aesthetically suitable for use in a consumer product.Tu, et al., U.S. Pat. No. 3,122,599 describes a leather-like sheet madefrom ground animal skin or leather which contains collagen fibers andsoluble collagen as well as other components derived from the animalskin. Tu discloses treating this mixture with chromium, dehydrating itwith acetone, and treating with oleic acid to produce a leather-likeproduct containing collagen fiber masses. Tu does not show that thesheet is compositionally, physically or aesthetically suitable for usein a consumer product. Tu, et al., U.S. Pat. No. 3,136,682 describes aprocess of making a leather-like material that contains a mixture ofcollagen fibers and a binder of water-soluble proteinaceous materialderived from animal skin. It also describes the use of a chromiumtanning agent and treatment with oleic acid. Tu describes a sheet ofgood appearance and feel, but does not show that it is suitable forincorporation into a consumer product. These products incorporatecoarse, ground or digested collagen fibers.

Cultured leather products. These products generally comprise a pluralityof layers containing collagen produced by culturing cells in vitro aredescribed by Forgacs, et al., U.S. 2016/0097109 A1 and by Greene, U.S.Pat. No. 9,428,817 B2. These products are produced in vitro bycultivation of cell explants or cultured collagen-producing cells. Suchcells produce and process collagen into quaternary bundles of collagenfibrils and do not have the random, non-antistrophic structure of thecollagen fibrils of the invention. Forgacs describes engineered animalskins, which may be shaped, to produce a leather product. Greendescribes a variety of products, such as footwear, apparel and luggagethat may incorporate leather that is cultured in vitro. US 2013/0255003describes producing collagen for leather-like products by growing bovineskin cells in culture. Other types of host cells have been utilized toproduce collagen for medical implants or to produce gelatin. Forexample, U.S. Patent Application US 2004/0018592 describes a way toproduce gelatin by recombinantly expressing bovine collagen in hostcells, such as yeast.

Medical products. Networks of collagen have been produced in vitro asmaterials for biomedical applications. In those applications, monomersof the collagen triple helix are extracted from animal tissue, such asbovine dermis, either by acid treatment or treatment with proteindegrading enzymes such as pepsin, to solubilize collagen from thetissue. Once purified, these solubilized collagens (often mixtures ofmonomers, dimers and trimers of the collagen triple helix) can befibrillated into fibrils through a pH shift in aqueous buffers. Underthe right conditions, the collagen monomers self-assemble into fibrils,and depending on their source and how they were isolated, the fibrilscan physically crosslink to form a solid hydrogel. In addition,recombinant collagens and collagen-like proteins have been shown tofibrillate in vitro through similar adjustments in pH and saltconcentration. Examples of such products for medical applicationsinclude a biodegradable collagen matrix made from a collagen slurry thatself-assembles into macroscopic collagen fibers, U.S. Pat. No.9,539,363, and an organized array of collagen fibrils produced by use ofexternal guidance structures or internal templates and the applicationof tension, U.S. Pat. No. 9,518,106. Collagen products used in medicine,such as for tissue engineering or grafting, often aim to providecollagen in a form similar to that in a particular tissue beingengineered or repaired. While fibrillation of soluble collagens andcollagen-like proteins has been explored to produce collagen hydrogelsfor biomedical applications, this technology has not been successfullyapplied to the production of a material having the strength andaesthetic properties of natural leather.

Synthetic plastic-based leathers. Attempts to create synthetic leatherhave come up short in reproducing leather's unique set of functional andaesthetic properties. Examples of synthetic leather materials includeClarino, Naugahyde®, Corfam, and Alcantara, amongst others. They aremade of various chemical and polymer ingredients, including polyvinylchloride, polyurethane, nitrocellulose coated cotton cloth, polyester,or other natural cloth or fiber materials coated with a syntheticpolymer. These materials are assembled using a variety of techniques,often drawing from chemical and textile production approaches, includingnon-woven and advanced spinning processes. While many of these materialshave found use in footwear, upholstery, and apparel applications, theyhave fallen short for luxury application, as they cannot match thebreathability, performance, hand feel, or aesthetic properties that makeleather so unique and beloved. To date, no alternative commercialleather-like materials have been made from a uniform network of collagenor collagen-like proteins. Synthetic plastic materials lack the chemicalcomposition and structure of a collagen network that produces anacceptable leather aesthetic. Unlike, synthetics, the chemicalcomposition of amino acid side groups along the collagen polypeptidechain, along with its organization into a strong yet porous, fibrousarchitecture allow stabilization and functionalization of the fibrilnetwork through crosslinking processes to produce the desirablestrength, softness and aesthetic of leather.

While fibrillation of soluble collagens and collagen-like proteins hasbeen explored to bind together ground or comminuted leather scraps orfor the production of collagen hydrogels for biomedical applications,harnessing this phenomenon to produce a commercially acceptableleather-like material has not been achieved.

In view of the problems with prior art natural leathers, and composite,cultured, and synthetic, plastic-based leather products the inventorsdiligently pursued a way to provide a biofabricated leather havingsuperior strength and uniformity and non-anisotropic properties thatincorporated natural components found in leather.

Described herein are materials composed of collagen fibrils fibrillatedin vitro that have leather-like properties imparted by crosslinking,dehydration and lubrication. Compared to tanned and fatliquored animalhides, these biofabricated materials can have structural, compositionaland functional uniformity, for example, advantageous substantiallynon-anisotropic strength and other mechanical properties as well as atop grain like aesthetic on both their top and bottom surfaces.

SUMMARY OF THE INVENTION

Among its other embodiments, the invention is directed to abiofabricated material composed of a network of crosslinked andlubricated collagen fibrils. It may be produced from collagen isolatedfrom an animal source or recombinant collagen. It can be produced fromcollagens that contain substantially no residues. Preferably it issubstantially free of large bundles of collagen fibers or othernon-hydroxylysine collagen components of leather, such as elastin. Thismaterial is composed of collagen which is also a major component ofnatural leather and is produced by a process of fibrillation of collagenmolecules into fibrils, crosslinking the fibrils and lubricating thecrosslinked fibrils. Unlike natural leathers, this biofabricatedmaterial exhibits non-anisotropic (not directionally dependent) physicalproperties, for example, a sheet of biofabricated material can havesubstantially the same elasticity or tensile strength when measured indifferent directions. Unlike natural leather, it has a uniform texturethat facilitates uniform uptake of dyes and coatings. Aesthetically, itproduces a uniform and consistent grain for ease of manufacturability.It can have substantially identical grain, texture and other aestheticproperties on both sides unlike natural leathers where the grainincreases from one side (e.g., distal surface) to the other (proximalinner layers).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing the composition of collagen in ahierarchical fashion. Reference character (1) shows each triple helicalcollagen monomer and how they are assembled with respect to neighboringcollagen monomers; (2) shows assembled collagen that makes up bandedcollagen fibrils; (3) shows the collagen fibrils at larger scale; (4)shows collagen fibrils aligned into fibers; and (5) shows bundles ofcollagen fibers.

FIG. 2A is a picture showing the composition of buffalo hide. The topgrain layer and the corium layer underneath are shown and the relativedegrees of higher order organization from collagen fibrils to collagenfiber bundles are indicated. The top grain layer is mostly composed offine collagen fibrils while the corium layer is mostly composed ofcoarser collagen fibers and fiber bundles.

FIGS. 2B and 2C compare the textures and grains of the outer and innersurfaces of a leather depicting fine grain on one side and coarsercorium on the other.

FIG. 3A is a scanning electron micrograph of the fibrillated collagenhydrogel showing a network of fine collagen fibrils.

FIG. 3B is a scanning electron micrograph of bovine corium showingcoarser fiber bundles.

FIG. 4 is a transmission electron micrograph of a fibrillated collagennetwork or hydrogel showing fibril banding.

DETAILED DESCRIPTION OF THE INVENTION

“Biofabricated material” or “biofabricated leather” as used herein is amaterial produced from collagen or a collagen-like protein. It can beproduced from non-human collagens such as bovine, buffalo, ox, dear,sheep, goat, or pig collagen, which may be isolated from a naturalsource like animal hide, by in vitro culture of mammalian or animalcells, recombinantly produced or chemically synthesized. It is not aconventional material or leather which is produced from animal skins.Methods for producing this biofabricated material or biofabricatedleather are disclosed herein and usually involve fibrillating anisolated or purified solution or suspension of collagen molecules toproduce collagen fibrils, crosslinking the fibrils, dehydrating thefibrils and lubricating the fibrils.

In contrast to natural leathers which exhibit heterogeneous internalcollagen structures, a biofabricated material or biofabricated leathercan exhibit a substantially uniform internal structure characterized byunbundled and randomly-oriented collagen fibrils throughout its volume.

The resulting biofabricated material may be used in any way that naturalleather is used and may be grossly similar in appearance and feel toreal leather, while having compositional, functional or aestheticfeatures that differentiate it from ordinary leather. For example,unlike natural leather, a biofabricated leather need not containpotentially allergenic non-collagen proteins or components found in anatural leather, a biofabricated leather may exhibit a similarflexibility and strength in all directions (non-anisotropy) due tosubstantial non-alignment of its collagen fibrils, and aesthetically mayhave a smooth grain texture on both sides. A biofabricated leather canexhibit uniformity of properties including uniform thickness andconsistency, uniform distribution of lubricants, crosslinkers and dyes,uniform non-anisotropic strength, stretch, flexibility and resistance topiping (or the tendency for natural leather to separate or splitparallel to a plane of a sheet). By selecting the content of collagenand processing conditions, biofabricated leather can be “tuned” to aparticular thickness, consistency, flexibility, softness, drape. surfacetexture or other functionality. Laminated, layered or composite productsmay comprise a biofabricated leather.

The term “collagen” refers to any one of the known collagen types,including collagen types I through XX, as well as to any othercollagens, whether natural, synthetic, semi-synthetic, or recombinant.It includes all of the collagens, modified collagens and collagen-likeproteins described herein. The term also encompasses procollagens andcollagen-like proteins or collagenous proteins comprising the motif(Gly-X-Y)n where n is an integer. It encompasses molecules of collagenand collagen-like proteins, trimers of collagen molecules, fibrils ofcollagen, and fibers of collagen fibrils. It also refers to chemically,enzymatically or recombinantly-modified collagens or collagen-likemolecules that can be fibrillated as well as fragments of collagen,collagen-like molecules and collagenous molecules capable of assemblinginto a nanofiber.

In some embodiments, amino acid residues, such as lysine and proline, ina collagen or collagen-like protein may lack hydroxylation or may have alesser or greater degree of hydroxylation than a corresponding naturalor unmodified collagen or collagen-like protein. In other embodiments,amino acid residues in a collagen or collagen-like protein may lackglycosylation or may have a lesser or greater degree of glycosylationthan a corresponding natural or unmodified collagen or collagen-likeprotein.

The collagen in a collagen composition may homogenously contain a singletype of collagen molecule, such as 100% bovine Type I collagen or 100%Type III bovine collagen, or may contain a mixture of different kinds ofcollagen molecules or collagen-like molecules, such as a mixture ofbovine Type I and Type III molecules. Such mixtures may include >0%, 10,20, 30, 40, 50, 60, 70, 80, 90, 95, 99 or <100% of the individualcollagen or collagen-like protein components. This range includes allintermediate values. For example, a collagen composition may contain 30%Type I collagen and 70% Type III collagen, or may contain 33.3% of TypeI collagen, 33.3% of Type II collagen, and 33.3% of Type III collagen,where the percentage of collagen is based on the total mass of collagenin the composition or on the molecular percentages of collagenmolecules.

“Collagen fibrils” are nanofibers composed of tropocollagen (triplehelices of collagen molecules). Tropocollagens also includetropocollagen-like structures exhibiting triple helical structures. Thecollagen fibrils of the invention may have diameters ranging from 1 nmand 1 μm. For example, the collagen fibrils of the invention may have anaverage or individual fibril diameter ranging from 1, 2, 3, 4, 5, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, or 1,000 nm (1 μm). This range includes all intermediatevalues and subranges. In some of the embodiments of the inventioncollagen fibrils will form networks, for example, as depicted by FIGS. 3and 4. Collagen fibrils can associate into fibrils exhibiting a bandedpattern as shown in FIG. 1 and these fibrils can associate into largeraggregates of fibrils. In some embodiments the collagen or collagen-likefibrils will have diameters and orientations similar to those in the topgrain or surface layer of a bovine or other conventional leather. Inother embodiments, the collagen fibrils may have diameters comprisingthe top grain and those of a corium layer of a conventional leather.

A “collagen fiber” is composed of collagen fibrils that are tightlypacked and exhibit a high degree of alignment in the direction of thefiber as shown in FIG. 1. It can vary in diameter from more than 1 μm tomore than 10 μm, for example >1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 μmor more. Some embodiments of the network of collage fibrils of theinvention do not contain substantial content of collagen fibers havingdiameters greater than 5 μm As shown in FIG. 2, the composition of thegrain surface of a leather can differ from its more internal portions,such as the corium which contains coarser fiber bundles.

“Fibrillation” refers to a process of producing collagen fibrils. It maybe performed by raising the pH or by adjusting the salt concentration ofa collagen solution or suspension. In forming the fibrillated collagen,the collagen may be incubated to form the fibrils for any appropriatelength of time, including between 1 min and 24 hrs and all intermediatevalues.

The fibrillated collagen described herein may generally be formed in anyappropriate shape and/or thickness, including flat sheets, curvedshapes/sheets, cylinders, threads, and complex shapes. These sheets andother forms may have virtually any linear dimensions including athickness, width or height greater of 10, 20, 30, 40, 50, 60, 70,80, 90mm; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 500, 1,000, 1,500,2,000 cm or more.

The fibrillated collagen in a biofabricated leather may lack any or anysubstantial amount of higher order structure. In a preferred embodiment,the collagen fibrils in a biofabricated leather will be unbundled andnot form the large collagen fibers found in animal skin and provide astrong and uniform non-anisotropic structure to the biofabricatedleather.

In other embodiments, some collagen fibrils can be bundled or alignedinto higher order structures. Collagen fibrils in a biofabricatedleather may exhibit an orientation index ranging from 0, >0, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, <1.0, or 1.0, wherein an orientationindex of 0 describes collagen fibrils that lack alignment with otherfibrils and an orientation index of 1.0 describes collagen fibrils thatare completely aligned. This range includes all intermediate values andsubranges. Those of skill in the art are familiar with the orientationindex which is also incorporated by reference to Sizeland, et al., J.Agric. Food Chem. 61: 887-892 (2013) or Basil-Jones, et al., J. Agric.Food Chem. 59: 9972-9979 (2011).

The methods disclosed herein make it possible to produce a biofabricatedleather comprising collagen fibrils differing in diameter from thoseproduced by an animal expressing the same type of collagen. Thecharacteristics of natural collagens, such as fibril diameter and degreeof crosslinking between fibrils are affected by genetic andenvironmental factors such as the species or breed of the animal and bythe condition of the animal , for example the amount of fat, type offeed (e.g. grain, grass), and level of exercise.

A biofabricated leather may be fibrillated and processed to containcollagen fibrils that resemble or mimic the properties of collagenfibrils produced by particular species or breeds of animals or byanimals raised under particular conditions.

Alternatively, fibrillation and processing conditions can be selected toprovide collagen fibrils distinct from those found in nature, such as bydecreasing or increasing the fibril diameter, degree of alignment, ordegree of crosslinking compared to fibrils in natural leather.

A crosslinked network of collagen, sometimes called a hydrogel, may beformed as the collagen is fibrillated, or it may form a network afterfibrillation; in some variations, the process of fibrillating thecollagen also forms gel-like network. Once formed, the fibrillatedcollagen network may be further stabilized by incorporating moleculeswith di-, tri-, or multifunctional reactive groups that includechromium, amines, carboxylic acids, sulfates, sulfites, sulfonates,aldehydes, hydrazides, sulfhydryls, diazarines, aryl-, azides,acrylates, epoxides, or phenols.

The fibrillated collagen network may also be polymerized with otheragents (e.g. polymers that are capable of polymerizing or other suitablefibers), which could be used to further stabilize the matrix and providethe desired end structure. Hydrogels based upon acrylamides, acrylicacids, and their salts may be prepared using inverse suspensionpolymerization. Hydrogels described herein may be prepared from polarmonomers. The hydrogels used may be natural polymer hydrogels, syntheticpolymer hydrogels, or a combination of the two. The hydrogels used maybe obtained using graft polymerization, crosslinking polymerization,networks formed of water soluble polymers, radiation crosslinking, andso on. A small amount of crosslinking agent may be added to the hydrogelcomposition to enhance polymerization.

Average or individual collagen fibril length may range from 100, 200,300, 400, 500, 600, 700, 800, 900, 1,000 (1 μm); 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 μm (1mm) throughout the entire thickness of a biofabricated leather. Theseranges include all intermediate values and subranges.

Fibrils may align with other fibrils over 50, 100, 200, 300, 400, 500 μmor more of their lengths or may exhibit little or no alignment. In otherembodiments, some collagen fibrils can be bundled or aligned into higherorder structures.

Collagen fibrils in a biofabricated leather may exhibit an orientationindex ranging from 0, >0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,<1.0, or 1.0, wherein an orientation index of 0 describes collagenfibrils that lack alignment with other fibrils and an orientation indexof 1.0 describes collagen fibrils that are completely aligned. Thisrange includes all intermediate values and subranges. Those of skill inthe art are familiar with the orientation index which is alsoincorporated by reference to Sizeland, et al., J. Agric. Food Chem. 61:887-892 (2013) or Basil-Jones, et al., J. Agric. Food Chem. 59:9972-9979 (2011).

Collagen fibril density of a biofabricated leather may range from about1 to 1,000 mg/cc, preferably from 5 to 500 mg/cc including allintermediate values, such as 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 and 1,000mg/cc.

The collagen fibrils in a biofabricated leather may exhibit a unimodal,bimodal, trimiodal, or multimodal distribution, for example, abiofabricated leather may be composed of two different fibrilpreparations each having a different range of fibril diameters arrangedaround one of two different modes. Such mixtures may be selected toimpart additive, synergistic or a balance of physical properties on abiofabricated leather conferred by fibrils having different diameters.

Natural leather products may contain 150-300 mg/cc collagen based on theweight of the leather product. A biofabricated leather may contain asimilar content of collagen or collagen fibrils as conventional leatherbased on the weight of the biofabricated leather, such as a collagenconcentration of 100, 150, 200, 250, 300 or 350 mg/cc.

The fibrillated collagen, sometimes called a hydrogel, may have athickness selected based on its ultimate use. Thicker or moreconcentrated preparations of the fibrillated collagen generally producethicker biofabricated leathers. The final thickness of a biofabricatedleather may be only 10, 20, 30, 40, 50, 60, 70, 80 or 90% that of thefibril preparation prior to shrinkage caused by crosslinking,dehydration and lubrication.

“Crosslinking” refers to formation (or reformation) of chemical bondswithin between collagen molecules. A crosslinking reaction stabilizesthe collagen structure and in some cases forms a network betweencollagen molecules. Any suitable crosslinking agent known in the art canbe used including, without limitation, mineral salts such as those basedon chromium, formaldehyde, hexamethylene diisocyanate, glutaraldehyde,polyepoxy compounds, gamma irradiation, and ultraviolet irradiation withriboflavin. The crosslinking can be performed by any known method; see,e.g., Bailey et al., Radiat. Res. 22:606-621 (1964); Housley et al.,Biochem. Biophys. Res. Commun. 67:824-830 (1975); Siegel, Proc. Natl.Acad. Sci. U.S.A. 71:4826-4830 (1974); Mechanic et al., Biochem.Biophys. Res. Commun. 45:644-653 (1971); Mechanic et al., Biochem.Biophys. Res. Commun. 41:1597-1604 (1970); and Shoshan et al., Biochim.Biophys. Acta 154:261-263 (1968) each of which is incorporated byreference.

Crosslinkers include isocyantes, carbodiimide, poly(aldehyde),poly(azyridine), mineral salts, poly(epoxies), enzymes, thiirane,phenolics, novolac, resole as well as other compounds that havechemistries that react with amino acid side chains such as lysine,arginine, aspartic acid, glutamic acid, hydroxylproline, orhydroxylysine.

A collagen or collagen-like protein may be chemically modified topromote chemical and/or physical crosslinking between the collagenfibrils. Chemical crosslinking may be possible because reactive groupssuch as lysine, glutamic acid, and hydroxyl groups on the collagenmolecule project from collagen's rod-like fibril structure. Crosslinkingthat involve these groups prevent the collagen molecules from slidingpast each other under stress and thus increases the mechanical strengthof the collagen fibers. Examples of chemical crosslinking reactionsinclude but are not limited to reactions with the s-amino group oflysine, or reaction with carboxyl groups of the collagen molecule.Enzymes such as transglutaminase may also be used to generate crosslinksbetween glutamic acid and lysine to form a stable γ-glutamyl-lysinecrosslink. Inducing crosslinking between functional groups ofneighboring collagen molecules is known in the art. Crosslinking isanother step that can be implemented here to adjust the physicalproperties obtained from the fibrillated collagen hydrogel-derivedmaterials.

Still fibrillating or fibrillated collagen may be crosslinked orlubricated. Collagen fibrils can be treated with compounds containingchromium or at least one aldehyde group, or vegetable tannins prior tonetwork formation, during network formation, or network gel formation.Crosslinking further stabilizes the fibrillated collagen leather. Forexample, collagen fibrils pre-treated with acrylic polymer followed bytreatment with a vegetable tannin, such as Acacia Mollissima, canexhibit increased hydrothermal stability. In other embodiments,glyceraldehyde may be used as a cross-linking agent to increase thethermal stability, proteolytic resistance, and mechanicalcharacteristics, such as Young's modulus and tensile stress, of thefibrillated collagen.

A biofabricated material containing a network of collagen fibrils maycontain 0, >0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20% or more of acrosslinking agent including tanning agents used for conventionalleather. The crosslinking agents may be covalently bound to the collagenfibrils or other components of a biofabricated material ornon-covalently associated with them. Preferably, a biofabricated leatherwill contain no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% of acrosslinking agent.

“Lubricating” describes a process of applying a lubricant, such as a fator other hydrophobic compound or any material that modulates or controlsfibril-fibril bonding during dehydration to leather or to biofabricatedproducts comprising collagen. A desirable feature of the leatheraesthetic is the stiffness or hand of the material. In order to achievethis property, water-mediated hydrogen bonding between fibrils and/orfibers is limited in leather through the use of lubricants. Examples oflubricants include fats, biological, mineral or synthetic oils, cod oil,sulfonated oil, polymers, organofunctional siloxanes, and otherhydrophobic compounds or agents used for fatliquoring conventionalleather as well as mixtures thereof. While lubricating is in some waysanalogous to fatliquoring a natural leather, a biofabricated product canbe more uniformly treated with a lubricant due to its method ofmanufacture, more homogenous composition and less complex composition.

Other lubricants include surfactants, anionic surfactants, cationicsurfactants, cationic polymeric surfactants, anionic polymericsurfactants, amphiphilic polymers, fatty acids, modified fatty acids,nonionic hydrophilic polymers, nonionic hydrophobic polymers, polyacrylic acids, poly methacrylic, acrylics, natural rubbers, syntheticrubbers, resins, amphiphilic anionic polymer and copolymers, amphiphiliccationic polymer and copolymers and mixtures thereof as well asemulsions or suspensions of these in water, alcohol, ketones, and othersolvents.

Lubricants may be added to a biofabricated material containing collagenfibrils. Lubricants may be incorporated in any amount that facilitatesfibril movement or that confers leather-like properties such asflexibility, decrease in brittleness, durability, or water resistance. Alubricant content can range from about 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60% by weight ofthe biofabricated leather.

“Dehydrating” or “dewatering” describes a process of removing water froma mixture containing collagen fibrils and water, such as an aqueoussolution, suspension , gel, or hydrogel containing fibrillated collagen.Water may be removed by filtration, evaporation, freeze-drying, solventexchange, vacuum-drying, convection-drying, heating, irradiating ormicrowaving, or by other known methods for removing water. In addition,chemical crosslinking of collagen is known to remove bound water fromcollagen by consuming hydrophilic amino acid residues such as lysine,arginine, and hydroxylysine among others. The inventors have found thatacetone quickly dehydrates collagen fibrils and may also remove waterbound to hydrated collagen molecules. Water content of a biofabricatedmaterial or leather after dehydration is preferably no more than 60% byweight, for example, no more than 5, 10, 15, 20, 30, 35, 40, 50 or 60%by weight of the biofabricated leather. This range includes allintermediate values. Water content is measured by equilibration at 65%relative humidity at 25° C. and 1 atm.

“Grain texture” describes a leather-like texture which is aestheticallyor texturally the similar to the texture of a full grain leather, topgrain leather, corrected grain leather (where an artificial grain hasbeen applied), or coarser split grain leather texture. Advantageously,the biofabricated material of the invention can be tuned to provide afine grain, resembling the surface grain of a leather such as thatdepicted by FIGS. 2A, 2B and 2C.

A “biofabricated leather product” includes products comprising at leastone component of a biofabricated leather such as foot ware, garments,gloves, furniture or vehicle upholstery and other leather goods andproducts. It includes but is not limited to clothing, such as overcoats,coats, jackets, shirts, trousers, pants, shorts, swimwear,undergarments, uniforms, emblems or letters, costumes, ties, skirts,dresses, blouses, leggings, gloves, mittens, foot ware, shoes, shoecomponents such as sole, quarter, tongue, cuff, welt, and counter, dressshoes, athletic shoes, running shoes, casual shoes, athletic, running orcasual shoe components such as toe cap, toe box, outsole, midsole,upper, laces, eyelets, collar, lining, Achilles notch, heel, andcounter, fashion or women's shoes and their shoe components such asupper, outer sole, toe spring, toe box, decoration, vamp, lining, sock,insole, platform, counter, and heel or high heel, boots, sandals,buttons, sandals, hats, masks, headgear, headbands, head wraps, andbelts; jewelry such as bracelets, watch bands, and necklaces; gloves,umbrellas, walking sticks, wallets, mobile phone or wearable computercoverings, purses, backpacks, suitcases, handbags, folios, folders,boxes, and other personal objects; athletic, sports, hunting orrecreational gear such as harnesses, bridles, reins, bits, leashes,mitts, tennis rackets, golf clubs, polo, hockey, or lacrosse gear,chessboards and game boards, medicine balls, kick balls, baseballs, andother kinds of balls, and toys; book bindings, book covers, pictureframes or artwork; furniture and home, office or other interior orexterior furnishings including chairs, sofas, doors, seats, ottomans,room dividers, coasters, mouse pads, desk blotters, or other pads,tables, beds, floor, wall or ceiling coverings, flooring; automobile,boat, aircraft and other vehicular products including seats, headrests,upholstery, paneling, steering wheel, joystick or control coverings andother wraps or coverings.

Many uses of leather products require a durable product that doesn't ripor tear, even when the leather has been stitched together. Typicalproducts that include stitched leather and require durable leatherinclude automobile steering wheel covers, automobile seats, furniture,sporting goods, sport shoes, sneakers, watch straps and the like. Thereis a need to increase the durability of biofabricated leather to improveperformance in these products. A biofabricated leather according to theinvention can be used to make any of these products.

Physical Properties of a biofabricated network of collagen fibrils or abiofabricated leather may be selected or tuned by selecting the type ofcollagen, the amount of concentration of collagen fibrillated, thedegree of fibrillation, crosslinking, dehydration and lubrication.

Many advantageous properties are associated with the network structureof the collagen fibrils which can provide strong, flexible andsubstantially uniform properties to the resulting biofabricated materialor leather. Preferable physical properties of the biofabricated leatheraccording to the invention include a tensile strength ranging from 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more MPa, a flexibilitydetermined by elongation at break ranging from 1, 5, 10, 15, 20, 25, 30%or more, softness as determined by ISO 17235 of 4, 5, 6, 7, 8 mm ormore, a thickness ranging from 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3. 1,4, 1,5, 1.6, 1.7, 1.8, 1.9, 2.0 mm or more, and acollagen density (collagen fibril density) of 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 mg/cc ormore, preferably 100-500 mg/cc. The above ranges include all subrangesand intermediate values.

Thickness. Depending on its ultimate application a biofabricatedmaterial or leather may have any thickness. Its thickness preferablyranges from about 0.05 mm to 20 mm as well as any intermediate valuewithin this range, such as 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 mm ormore. The thickness of a biofabricated leather can be controlled byadjusting collagen content.

Elastic modulus. The elastic modulus (also known as Young's modulus) isa number that measures an object or substance's resistance to beingdeformed elastically (i.e., non-permanently) when a force is applied toit. The elastic modulus of an object is defined as the slope of itsstress-strain curve in the elastic deformation region. A stiffermaterial will have a higher elastic modulus. The elastic modulus can bemeasured using a texture analyzer.

A biofabricated leather can have an elastic modulus of at least 100 kPA.It can range from 100 kPa to 1,000 MPa as well as any intermediate valuein this range, such as 0.1, 0.2, 0.3 , 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800,900, or 1,000 MPA. A biofabricated leather may be able to elongate up to300% from its relaxed state length, for example, by >0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or300% of its relaxed state length.

Tensile strength (also known as ultimate tensile strength) is thecapacity of a material or structure to withstand loads tending toelongate, as opposed to compressive strength, which withstands loadstending to reduce size. Tensile strength resists tension or being pulledapart, whereas compressive strength resists compression or being pushedtogether.

A sample of a biofabricated material may be tested for tensile strengthusing an Instron machine. Clamps are attached to the ends of the sampleand the sample is pulled in opposite directions until failure. Goodstrength is demonstrated when the sample has a tensile strength of atleast 1 MPa. A biofabricated leather can have a tensile strength of atleast 1 kPA. It can range from 1 kPa to 100 MPa as well as anyintermediate value in this range, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,50, 100, 200, 300, 400, 500 kPA; 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80,90, or 100 MPa.

Tear strength (also known as tear resistance) is a measure of how well amaterial can withstand the effects of tearing.More specifically howeverit is how well a material (normally rubber) resists the growth of anycuts when under tension, it is usually measured in kN/m. Tear resistancecan be measured by the ASTM D 412 method (the same used to measuretensile strength, modulusand elongation). ASTM D 624 can be used tomeasure the resistance to the formation of a tear (tear initiation) andthe resistance to the expansion of a tear (tear propagation). Regardlessof which of these two is being measured, the sample is held between twoholders and a uniform pulling force applied until the aforementioneddeformation occurs. Tear resistance is then calculated by dividing theforce applied by the thickness of the material. A biofabricated leathermay exhibit tear resistance of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, 45, 50, 100, 150 or 200% more than that of aconventional top grain or other leather of the same thickness comprisingthe same type of collagen, e.g., bovine Type I or Type III collagen,processed using the same crosslinker(s) or lubricants. A biofabricatedmaterial may have a tear strength ranging from about 1 to 500 N, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425,450, 475, or 500 as well as any intermediate tear strength within thisrange.

Softness. ISO 17235:2015 specifies a non-destructive method fordetermining the softness of leather. It is applicable to all non-rigidleathers, e.g. shoe upper leather, upholstery leather, leather goodsleather, and apparel leather. A biofabricated leather may have asoftness as determined by ISO 17235 of 2, 3, 4, 5, 6, 7, 8, 10, 11, 12mm or more.

Grain. The top grain surface of leather is often regarded as the mostdesirable due to its soft texture and smooth surface. The top grain is ahighly porous network of collagen fibrils. The strength and tearresistance of the grain is often a limitation for practical applicationsof the top grain alone and conventional leather products are oftenbacked with corium having a much coarser grain. FIGS. 2A, 2B and 2Ccompare top grain and corium leather surfaces. A biofabricated materialas disclosed herein which can be produced with strong and uniformphysical properties or increased thickness can be used to provide topgrain like products without the requirement for corium backing.

Content of other components. In some embodiments, the collagen is freeof other leather components such as elastin or non-structural animalproteins. However, in some embodiments the content of actin, keratin,elastin, fibrin, albumin, globulin, mucin, mucinoids, noncollagenstructural proteins, and/or noncollagen nonstructural proteins in abiofabricated leather may range from 0, 1, 2, 3,4, 5, 6, 7, 8, 9 to 10%by weight of the biofabricated leather. In other embodiments, a contentof actin, keratin, elastin, fibrin, albumin, globulin, mucin, mucinoids,noncollagen structural proteins, and/or noncollagen nonstructuralproteins may be incorporated into a biofabricated leather in amountsranging from >0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20% or more by weight of a biofabricated leather. Suchcomponents may be introduced during or after fibrillation,cross-linking, dehydration or lubrication.

A “leather dye” refers to dyes which can be used to color leather orbiofabricated leather. These include acidic dyes, direct dyes, lakes,sulfur dyes, basic dyes and reactive dyes. Dyes and pigments can also beincorporated into a precursor of a biofabricated leather, such as into asuspension or network gel comprising collagen fibrils during productionof the biofabricated leather.

“Fillers”. In some embodiments a biofabricated leather may comprisefillers, other than components of leather, such as microspheres. One wayto control the organization of the dehydrated fibril network is toinclude filling materials that keep the fibrils spaced apart duringdehydration. These filler materials include nanoparticles,microparticles, or various polymers such as syntans commonly used in thetanning industry. These filling materials could be part of the finaldehydrated leather material, or the filling materials could besacrificial, that is they are degraded or dissolved away leaving openspace for a more porous fibril network. The shape and dimension of thesefillers may also be used to control the orientation of the dehydratedfibril network.

In some embodiments a filler may comprise polymeric microsphere(s),bead(s), fiber(s), wire(s), or organic salt(s). Other materials may alsobe embedded or otherwise incorporated into a biofabricated leather orinto a network of collagen fibrils according to the invention. Theseinclude, but are not limited to one fibers, including both woven andnonwoven fibers as well as cotton, wool, cashmere, angora, linen,bamboo, bast, hemp, soya, seacell, fibers produced from milk or milkproteins, silk, spider silk, other peptides or polypeptides includingrecombinantly produced peptides or polypeptides, chitosan, mycelium,cellulose including bacterial cellulose, wood including wood fibers,rayon, lyocell, vicose, antimicrobial yarn (A.M.Y.), Sorbtek, nylon,polyester, elastomers such as lycra®, spandex or elastane and otheripolyester-polyurethane copolymers, aramids, carbon including carbonfibers and fullerenes, glass including glass fibers and nonwovens,silicon and silicon-containing compounds, minerals, including mineralparticles and mineral fibers, and metals or metal alloys, includingthose comprising iron, steel, lead, gold, silver, platinum, copper, zincand titanium, which may be in the form of particles, fibers, wires orother forms suitable for incorporating into biofabricated leather. Suchfillers may include an electrically conductive material, magneticmaterial, fluorescent material, bioluminescent material, phosphorescentmaterial or other photoluminescent material, or combinations thereof.Mixtures or blends of these components may also be embedded orincorporated into a biofabricated leather, for example, to modify thechemical and physical properties disclosed herein.

Method of making. A method of forming biofabricated leather fromcollagen includes the steps of fibrillating, crosslinking,dehydrating/dewatering and lubricating in any order. For example, acollagen solution may be fibrillated, the fibrils may be crosslinkedwith an agent such as glutaraldehyde, then coated with a lubricant suchas a sulfited oil, and then dehydrated through filtration to form afibrillated collagen leather. However, the method of making is notlimited to this particular order of steps.

Alternatively, following fibril crosslinking, the fibrils can bedehydrated through a solvent exchange with acetone, followed by fatliquoring with a sulfited oil before evaporating away the solvent toform a fibrillated collagen leather. In addition, the incorporation ofchemical or physical crosslinks between fibrils (to impart materialstrength) can be accomplished at any point during the process. Forexample, a solid fibrillated collagen, sometimes called a hydrogel, canbe formed, then this fibril network can be dehydrated through a solventexchange with acetone, followed by fat liquoring with a sulfited oil.Further, the collagen fibrils can be crosslinked into a network throughthe incorporation of other polymers such as those typically used inresin formulations.

Materials such as lubricants, humectants, dyes and other treating agentscan be uniformly distributed through a biofabricated leather productduring the biofabrication process This is an advantage compared toconventional leather tanning and fat liquoring which due to itsstructural heterogeneity often makes uniform treatment impossible.Further, as chemical agents can be incorporated before networkformation, smaller amounts of treatment chemicals would be necessary asthere is reduced chemical loss by not having to penetrate a collagennetwork from a float containing the treatment chemicals. Unlike hightemperatures often used to treat natural leather, a biofabricated can beheated at ambient temperature or at a temperature no greater than 37° C.during processing before evaporating away the solvent to form afibrillated collagen leather. Alternatively, collagen fibrils can becrosslinked and lubricated in suspension before forming a networkbetween fibrils during dehydration or through the addition of a bindingagent to the suspension or to the dehydrated material.

A method of forming a biofabricated leather material may includeinducing fibrillation of collagen in a solution; crosslinking (e.g.,tanning) and dehydrating the fibrillated collagen, which may appear inthe form of a hydrogel, to obtain a fibrillated collagen sheet or otherproduct, and incorporating at least one humectant or lubricant, such asa fat or oil into the fibrillated collagen sheet or product to obtain aflexible biofabricated leather.

A method of biofabricating a leather from fibrils may include inducingfibrillation of collagen or collagen-like proteins in a solution toobtain a fibrillated collagen hydrogel; crosslinking the fibrillatedcollagen hydrogel to obtain a fibrillated collagen hydrogel leather; andincorporating at least one lubricating oil into the fibrillated collagenhydrogel leather.

In the processes described herein for producing a biofabricated leather,the order of the steps for forming biofabricated leather may be variedor two or more steps may be performed simultaneously. For example,fibrillating and crosslinking may be performed together or by additionof one or more agents, or crosslinker and lubricant may be incorporatedin the solution prior to fibrillating the collagen, etc.

The collagen or collagen-like proteins may be obtained throughextraction of collagen from an animal source, such as, but not limitedto bovine hide or tendon collagen extraction. Alternatively, thecollagen or collagen-like proteins may be obtained from a non-animalsource, for example through recombinant DNA techniques, cell culturetechniques, or chemical peptide synthesis.

Any of these methods may include polymerizing the collagen orcollagen-like proteins into dimers, trimers, and higher order oligomersprior to fibrillation, and/or chemically modifying the collagen orcollagen-like proteins to promote crosslinking between the collagen orcollagen-like proteins.

Any of these methods may include functionalizing the collagen orcollagen-like proteins with one or a combination of chromium, amine,carboxylic acid, sulfate, sulfite, sulfonate, aldehyde, hydrazide,sulfhydryl, diazirine, aryl, azide, acrylate, epoxide, or phenol group.

Inducing fibrillation may include adding a salt or a combination ofsalts, for example, the salt or combination of salts may include:Na₃PO₄, K₃PO₄, KCl, and NaCl, the salt concentration of each salt may bebetween 10 mM to 5M, etc.

In general, inducing fibrillation may comprise adjusting the pH with anacid or a base, adding a nucleating agent, such as a branched collagenmicrogel, wherein the nucleating agent has a concentration between 1 mMto 100 mM.

The fibrillated collagen may be stabilized with a chromium compound, analdehyde compound, or vegetable tannins, or any other crosslinkingagent. For example, the fibrillated collagen may be stabilized with achromium compound, an aldehyde compound, or vegetable tannins, whereinthe chromium, aldehyde, or vegetable tannin compounds having aconcentration of between 1 mM to 100 mM.

Any of these methods may include adjusting the water content of thefibrillated collagen to 5, 10, 20, 25, 30, 40, 50 or 60% or less byweight to obtain the fibrillated collagen hydrogel leather. For example,the fibrillated collagen material may be dehydrated. Any of thesemethods may also include dyeing and/or applying a surface finish to thefibrillated collagen leather.

The selection of collagen starting materials for biofabricating theengineered leather materials described herein can be controlled, theresulting product may differential formed with physical and aestheticproperties for distinct end uses, such as with features useful infootware and different features useful in apparel. In general, thebiofabricated fibrillated collagen hydrogel-derived leathers describedherein are formed from solutions of collagen that are induced toself-assemble into collagen fibrils.

The collagen fibrils, unlike endogenous collagen fibrils, are notassembled into any high-order structures (e.g., bundles of fibers), butremain somewhat disordered, more particularly unbundled fibrils. Whenassembled in vivo, collagen fibrils are typically aligned laterally toform bundles having a higher order of structure and make up tough,micron-sized collagen fibers found, e.g., in skin. A characteristicfeature of native collagen fibrils is their banded structure. Thediameter of the native fibril changes slightly along the length, with ahighly reproducible D-band repeat of approximately 67 nm. In some of themethods described herein, collagen fibrils may be unbanded and unbundledor may be banded and unbundled or may have a D-band of different spacingranging from 1 to 100 nm and all intermediate values in this range). Thecollagen fibrils may be randomly oriented (e.g., un-oriented or notoriented in any particular direction or axis).

The starting material used to form the biofabricated leather material asdescribed herein may include any appropriate non-human collagen sourceor modified or engineered collagens that can be fibrillated.

Various forms of collagen are found throughout the animal kingdom. Thecollagen used herein may be obtained from animal sources, including bothvertebrates and invertebrates, or from synthetic sources. Collagen mayalso be sourced from byproducts of existing animal processing. Collagenobtained from animal sources may be isolated using standard laboratorytechniques known in the art, for example, Silva et. Al., Marine OriginCollagens and its Potential Applications, Mar. Drugs, 2014 December.,12(12); 5881-5901).

One major benefit of the biofabricated leather materials and methods forforming them described herein is that collagen may be obtained fromsources that do not require killing of an animal.

The collagen described herein also may be obtained by cell culturetechniques including from cells grown in a bioreactor.

Collagen may also be obtained via recombinant DNA techniques. Constructsencoding non-human collagen may be introduced into host organisms toproduce non-human collagen. For instance, collagen may also be producedwith yeast, such as Hansenula polymorpha, Saccharomyces cerevisiae,Pichia pastoris and the like as the host. Further, in recent years,bacterial genomes have been identified that provide the signature(Gly-Xaa-Yaa)n repeating amino acid sequence that is characteristic oftriple helix collagen. For example, gram positive bacteriumStreptococcus pyogenes contains two collagen-like proteins, Scl1 andScl2 that now have well characterized structure and functionalproperties. Thus, it would be possible to obtain constructs inrecombinant E. coli systems with various sequence modifications ofeither Scl1 or Scl2 for establishing large scale production methods.Collagen may also be obtained through standard peptide synthesistechniques. Collagen obtained from any of the techniques mentioned maybe further polymerized. Collagen dimers and trimers are formed fromself-association of collagen monomers in solution.

As an initial step in the formation of the collagen materials describedherein, the starting collagen material may be placed in solution andfibrillated. Collagen fibrillation may be induced through theintroduction of salts to the collagen solution. The addition of a saltor a combination of salts such as sodium phosphate, potassium phosphate,potassium chloride, and sodium chloride to the collagen solution maychange the ionic strength of the collagen solution. Collagenfibrillation may occur as a result of increasing electrostaticinteractions, through greater hydrogen bonding, Van der Waalsinteractions, and covalent bonding. Suitable salt concentrations mayrange, for example, from approximately 10 mM, 50 mM, 100 mM, 500 mM, 1M,2M, 3M, 4M to 5M as well as any intermediate value within this range.

Collagen fibrillation may also be induced or enhanced with a nucleatingagent other than salts. Nucleating agents provide a surface on whichcollagen monomers can come into close contact with each other toinitiate fibrillation or can act as a branch point in which multiplefibrils are connected through the nucleating agent. Examples of suitablenucleating agents include but are not limited to: microgels containingcollagen, collagen micro or nanoparticles, metallic particles ornaturally or synthetically derived fibers. Suitable nucleating agentconcentrations may range from approximately 1 mM to 100 mM.

A collagen network may also be highly sensitive to pH. During thefibrillation step, the pH may be adjusted to control fibril dimensionssuch as diameter and length. The overall dimensions and organization ofthe collagen fibrils will affect the toughness, stretch-ability, andbreathability of the resulting fibrillated collagen derived materials.This may be of use for fabricating fibrillated collagen derived leatherfor various uses that may require different toughness, flexibility, andbreathability. Adjustment of pH, with or without a change in saltconcentration may be used for fibrillation.

One way to control the organization of the dehydrated fibril network isto include filling materials that keep the fibrils spaced apart duringdrying. These filler materials could include nanoparticles,microparticles, or various polymers such as syntans commonly used in thetanning industry. These filling materials could be part of the finaldehydrated leather material, or the filling materials could besacrificial, that is they are degraded or dissolved away leaving openspace for a more porous fibril network.

The collagen or collagen-like proteins may be chemically modified topromote chemical and physical crosslinking between the collagen fibrils.Chemical crosslinking may be possible because reactive groups such aslysine, glutamic acid, and hydroxyl groups on the collagen moleculeproject from collagen's rod-like fibril structure. Crosslinking thatinvolve these groups prevent the collagen molecules from sliding pasteach other under stress and thus increases the mechanical strength ofthe collagen fibers. Examples of chemical crosslinking reactions includebut are not limited to reactions with the s-amino group of lysine, orreaction with carboxyl groups of the collagen molecule. Enzymes such astransglutaminase may also be used to generate crosslinks betweenglutamic acid and lysine to form a stable y-glutamyl-lysine crosslink.Inducing crosslinking between functional groups of neighboring collagenmolecules is known in the art. Crosslinking is another step that can beimplemented here to adjust the physical properties obtained from thefibrillated collagen hydrogel-derived materials.

Once formed, the fibrillated collagen network may be further stabilizedby incorporating molecules with di-, tri-, or multifunctional reactivegroups that include chromium, amines, carboxylic acids, sulfates,sulfites, sulfonates, aldehydes, hydrazides, sulfhydryls, diazarines,aryl-, azides, acrylates, epoxides, or phenols.

The fibrillated collagen network may also be polymerized with otheragents (e.g. polymers that are capable of polymerizing or other suitablefibers) that form a hydrogel or have fibrous qualities, which could beused to further stabilize the matrix and provide the desired endstructure. Hydrogels based upon acrylamides, acrylic acids, and theirsalts may be prepared using inverse suspension polymerization. Hydrogelsdescribed herein may be prepared from polar monomers. The hydrogels usedmay be natural polymer hydrogels, synthetic polymer hydrogels, or acombination of the two. The hydrogels used may be obtained using graftpolymerization, crosslinking polymerization, networks formed of watersoluble polymers, radiation crosslinking, and so on. A small amount ofcrosslinking agent may be added to the hydrogel composition to enhancepolymerization.

Any appropriate thickness of the fibrillated collagen hydrogel may bemade as described herein. Because the final thickness will be much less(e.g., between 10-90% thinner) than the hydrogel thickness, the initialhydrogel thickness may depend on the thickness of the final productdesired, presuming the changes to the thickness (or overall volume)including shrinkage during crosslinking, dehydration and/or adding oneor more oils or other lubricants as described herein.

A hydrogel thickness may be between 0.1 mm and 50 cm or any intermediatevalue within this range. In forming the fibrillated hydrogel, thehydrogel may be incubated to form the thickness for any appropriatelength of time, including between 1 min and 24 hrs.

The fibrillated collagen hydrogels described herein may generally beformed in any appropriate shape and/or thickness, including flat sheets,curved shapes/sheets, cylinders, threads, and complex shapes. Further,virtually any linear size of these shapes. For example, any of thesehydrogels may be formed into a sheet having a thickness as described anda length of greater than 10 mm (e.g., greater than, in cm, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1500, etc.) and widththat is greater than 10 mm, such as greater than, in cm, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 1100, 1200, 1500, etc.

Once the collagen fibrils, often characterized as a hydrogel, haveformed or during formation, they may be crosslinked. For example, thefibrillated collagen hydrogel be treated with compounds containingchromium or at least one aldehyde group, or vegetable tannins prior togel formation, during gel formation, or after gel formation, to furtherstabilize the fibrillated collagen hydrogel. For example, collagenfibrils may be pre-treated with acrylic polymer followed by treatmentwith a vegetable tannin (e.g., Acacia Mollissima) may exhibit increasedhydrothermal stability. In other examples, glyceraldehyde may be used asa cross-linking agent that may increase the thermal stability,proteolytic resistance, and mechanical characteristics (e.g. Young'smodulus, tensile stress) of the fibrillated collagen hydrogel.

Fibrillation of a solution of collagen as described herein (e.g.,formation of collagen fibrils that are lacking in higher orderorganization such as bundles) may be visually tracked. For example, asfibrillation is induced, the clear solution of collagen, which haslittle absorbance at 400 nm, becomes opaque and having an absorbance ofapproximately 0.5 at 400 nm. Depending on the temperature and volume ofstarting material, the fibrillation and hydrogel formation may occursomewhat quickly after induction and be largely complete after an hourand a half, as shown by the absorbance values leveling off after 70minute's time passing. An increase in storage modulus (or viscoelasticqualities of the material) of the fibrillated collagen hydrogel afterinduction from around 1 Pa (for the solution of collagen) toapproximately 400 Pa for the fibrillated collagen hydrogel may occur.

As mentioned above and illustrated in FIGS. 1 and 2, animal skintypically includes fibrils that are ordered into higher-orderstructures, including the presence of banding (having regular lacunarregions) and formation of multiple fibrils into aligned fibers which maythen bundled into collagen bundles. In contrast, the collagen hydrogelsand therefore the biofabricated leathers described herein may have aprimary disorganized collagen fibril structure throughout the entirethickness (in some cases entire volume) of the material. Specifically,the collagen structure of the biofabricated leathers formed fromcollagen hydrogels may be primarily unbundled and un-oriented along anyparticular axis. In some variations the collagen fibrils may be unbanded(e.g., greater than 10% unbanded, greater than 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, etc. unbanded throughout the volume). Furthermore, theorientation of the collagen fibrils within the volume (or throughout thevolume) may be un-oriented, or randomly oriented, and this lack oforientation may be the same throughout the volume, rather than changingthrough the volume thickness as in native leather, which may have changefrom bundles of collagen fibrils that are vertically oriented to bundlesthat are horizontally oriented in the thickness. Any of the propertieswhich are the same at any level thickness of the hydrogel and thereforeresulting leather material may be referred to herein as “uniformly” thesame throughout the thickness.

In addition, any of the biofabricated leathers described herein may havea uniform distribution of fibrils throughout the thickness of the geland therefore resulting leather material. This is in contrast withnative leathers, such as the material shown in FIG. 2, showing anincrease in the number of fiber bundles through the thickness of thematerial.

The lack of higher-level organization of the fibrillated collagenhydrogels and leather material formed from them is apparent in FIGS. 3Aand 3B. FIG. 3A shows a scanning electron micrograph of a fibrillatedcollagen hydrogel formed as described herein. Similarly, FIG. 4 shows atransmission electron micrograph through a fibrillated collagenhydrogel. The transmission electron micrograph and the scanning electronmicrograph both show the fibrillated collagen hydrogel as being adisordered tangle of collagen fibrils. As previously mentioned, thedensity and to some extent, the pattern of collagen fibril formation maybe controlled by adjusting the pH of the collagen solution duringfibrillation induction along with the concentration of fibrils duringdehydration. FIG. 3 also shows a scanning electron micrograph of bovinecorium. In comparison with a natural bovine corium (FIG. 3B), thefibrillated collagen network is much more random and lacks the apparentstriations. Although the overall size of the fibrils may be similar, thearrangement of these fibrils is quite different. Such ultrastructuraldifferences between the collagen fibrils within the fibrillated collagenhydrogel and natural tissue such as bovine corium (and resulting leathermade therefrom) may not be an issue in the final biofabricated leatherproduct may be as soft or softer, and more pliable than natural leather,and may have a similar appearance.

The fibrillated collagen, sometimes called a hydrogel, may then bedehydrated to rid the fibrillated collagen hydrogel of the majority ofits water content. Removing the water from the fibrillated collagenhydrogel may change its physical quality from a hydrated gel to apliable sheet. The material may be treated to prevent breakage/tearing.For example, care may be taken not to remove too much water from thefibrillated collagen. In some examples, it may be desirable to dehydratethe fibrillated collagen to have a water content of less than 5, 10, 15,20, 25, 30, 40, 50 or 60%. Water content is determined by equilibrationat 25° C. at 1 atm pressure at a relative humidity of 65%.

Dehydration may involve air drying, vacuum and pressure filtration,solvent exchange or the like. For example, fibrillated collagen hydrogelmay also undergo dehydration through replacement of its water contentwith organic solvents. Suitable organic solvents may include, but arenot limited to acetone, ethanol, diethyl ether, and so forth.Subsequently, the organic solvents may be evaporated (e.g. air drying,vacuum drying, etc.). It is also possible to perform successive steps ofdehydration using one or more than one organic solvent to fine tune thelevel of dehydration in the final product.

After or during dehydration, the fibrillated collagen material may betreated with lubricants and/or oils to impart greater flexibility andsuppleness to the fibrillated collagen material. Using a combination ofoil and solvent may allow the oil to better penetrate the fibrillatedcollagen network compared to using oil by itself. Oil by itself willonly likely penetrate the exposed surfaces but may not readilyinfiltrate the entire thickness of the fibrillated collagen material ina reasonable amount of time. Once the oil/solvent composition haspenetrated the entire thickness of the material, the solvent may then beremoved. In some embodiments, the resulting fibrillated collagenmaterial exhibits a more leather-like appearance as compared to thedehydrated fibrillated collagen material prior to lubricant and/or oiltreatment. Suitable oils and lubricants may include but are not limitedto castor oil, pine oil, lanolin, mink oil, neatsfoot oil, fish oil,shea butter, aloe, and so forth.

Lubricating the dehydrated and crosslinkled fibrillated collagen networkor hydrogel to form a leather material may result in a material havingproperties that are similar, or better, than the properties of naturalleather. The solutions that included a combination of oils and organicsolvent increased the mass and the softness (inversely proportional tothe slope of the stress-strain curve) of the dehydrated fibrillatedcollagen material. This is due to the combination of oils and organicsolvents penetrating the dehydrated fibrillated collagen material andonce penetrated through, the oils remained distributed throughout thematerial, while the organic solvents are able to evaporate away. Whilenot shown, the use of oils alone may not be as effective in penetratingentirely through the dehydrated fibrillated collagen material.

The resulting fibrillated collagen materials then may be treatedsimilarly to natural leather derived from animal hide or skin, and bere-tanned, dyed, and/or finished. Additional processing steps mayinclude: crosslinking, re-tanning, and surface coating. Crosslinking andre-tanning may include sub-processes such as wetting back (re-hydratingsemi-processed leather), sammying (45-55% water is squeezed from theleather), splitting (leather is split into one or more layers), shaving(leather is thinned), neutralization (pH of leather is adjusted tobetween 4.5 and 6.5), dyeing (leather is colored), fat liquoring (fats,oils, waxes are fixed to the leather fibers), filling (dense/heavychemicals to make leather harder and heavier), stuffing (fats, oils,waxes added between leather fibers), fixation (unbound chemicals arebonded/trapped and removed), setting (grain flatness are imparted andexcess water removed), drying (leather is dried to desired moisturelevels, 10-25%), conditioning (moisture is added to leather to a 18-28%level), softening (physical softening of leather by separating thefibers), or buffing (abrading surface of leather to reduce nap and graindefects). Surface coating may include any one or combination of thefollowing steps: oiling (leather coated with raw oil or oils), buffing,spraying, roller coating, curtain coating, polishing, plating,embossing, ironing, or glazing.

Unlike animal hides, where the hide has to be trimmed to obtain thedesired thickness or dimensions, the engineered leather material may befabricated with a wide range of thicknesses as well as the desireddimensions with a particular end product in mind.

The production of such engineered leather materials may also generateless waste by bypassing the step of removing excess proteins, fats, andhair necessary for treating natural animal hide in the leatherproduction process, which results in less environmental impact from thedisclosed process and the products derived from these methods.

Embodiments of the Invention

The invention includes, but is not limited to biofabricated materialshaving the features described below.

In a first embodiment, the invention is directed to a materialcomprising a network of collagen fibers, such as a biofabricatedmaterial or biofabricated leather:

(i) comprising a network of non-human collagen fibrils, wherein lessthan 5, 10, 15, 20, 25, 30, 35, or 40% by weight of the collagen fibrilsin the material are in the form of collagen fibers having a diameter of1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm or more and/or are in the form offibrils aligned for 100 μm or more of their lengths; wherein saidmaterial contains no more than 10, 20, 30, 40, 50, or 60% by weightwater; wherein said material contains at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, or 40% by weight of a lubricant; and wherein optionally,the material comprises a top and bottom surface or an inner and outersurface; or

(ii) comprising a network of recombinant non-human collagen fibrils,wherein the collagen contains substantially no 3-hydroxyproline, and,optionally, substantially no hydroxylysine; wherein said materialcontains no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60%by weight water; wherein the material contains at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, or 40% of a lubricant; and wherein optionally,the material comprises a top and bottom surface or an inner and outersurface. Water content in this material is preferably no more than 25 to40%. Lubricant content may be selected to match or not exceed theabsorptive capacity of the biofabricated material for a lubricant. Sucha material may comprise mammalian collagen, such as bovine Type I orType III collagen. Preferably it will not contain hair, hairfollicle(s), or fat(s) of an animal that naturally expresses thecollagen molecules it contains. For example, it may contain less than 1,2, 3, 4, 5, 6, 7, 8, 9, or 10% by weight of actin, keratin, elastin,fibrin, albumin, globulin, mucin, mucinoids, noncollagen structuralproteins, and/or noncollagen nonstructural proteins found inconventional leather. It may be substantially free of other collagenousproteins, carbohydrates, nucleic acids, or lipids, or immunogens,antigens, or allergens found in a conventional leather, such as ananimal that naturally expresses the collagen molecules in abiofabricated material. Alternative embodiments may incorporate 1, 2, 3,4, 5, 6, 7, 8, 9, or 10% of one or more of actin, keratin, elastin,fibrin, albumin, globulin, mucin, mucinoids, noncollagen structuralproteins, and/or noncollagen nonstructural proteins found inconventional leather.

The collagen used to produce the fibrils in this material may beisolated from a natural source, preferably in a purified form, or it maybe recombinantly produced or produced by chemical synthesis. It maydifferent in chemical structure from collagen obtained from a naturalsource, for example, if may contain a lower content of, or substantiallyno 3-hydroxyproline, and, optionally, substantially no hydroxylysine,glycosylated or crosslinked amino acid residues, or otherpost-translational modifications of a collagen amino acid sequence.Alternatively, it may contain a higher content of hydroxylated aminoacid residues, glycosylated residues, crosslinks or other chemicalmodifications.

The material described above generally comprises a network of collagenfibrils which may exhibit a fibril density of between 5, 10, 20, 50,100, 200, 300, 400, 500, 600, 700, 800, 900, and 1,000 mg/cc, preferablybetween 100 and 500 mg/cc. These fibrils or network of fibrils canconfer a grain texture, such as a top grain texture, feel, or aestheticon a biofabricated material or biofabricated leather. However, abiofabricated material can exhibit a porosity and other physicalproperties that are more uniform than a corresponding conventionalleather which can be controlled or tuned by control of composition,fibril size, crosslinking and lubricating in a biofabricated product.

In many embodiments, the biofabricated material described above willhave a top and bottom surface, or an inner and outer surface, comprisingthe collagen fibrils. One or more of these surfaces may be externallyexposed. A single layer of biofabricated material can exhibitsubstantially identical grain and appearance on both of its sides,unlike conventional leather products where collagen fibril or fiberdiameters increase for more inner layers of a hide.

In other embodiments a biofabricated material may be cast, molded orotherwise configured into a particular shape which can exhibitsubstantially uniform properties over its surface(s).

The collagen fibrils in a biofabricated material can be tuned to have aparticular diameter. The distribution of fibril diameters may exhibit asubstantially unimodal distribution, a bimodal distribution, a trimodaldistribution or other multimodal distributions. Multimodal distributionsmay be composed of two or more different preparations of fibrilsproduced using different fibrillation conditions. In a substantiallyunimodal distribution >50, 60, 70, 80, 90, 95 or 99% of diameters of thefibrils distribute around a single mode. In bimodal distributions atleast 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% of the fibrils willdistribute around one mode. In trimodal and other multimodaldistributions, generally, at least about 5, 10, 15, 20, 25, 30% or more(depending on the number of modes) of the fibril diameters willdistribute around a mode.

A biofabricated material may contain fibrils where at least 50, 60, 70,80, 90, 95, or 99% of the collagen fibrils have diameters between 1 nmand 1 μm. Fibril diameters may be determined by methods known in the artincluding by visual inspection of micrographs or electron micrographs,such as scanning or transmission electronmicrographs. For example, thecollagen fibrils may have a collective average or individual fibrildiameter ranging from 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80,90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 nm (1 μm).

The collagen fibrils in the biofabricated material described above areusually crosslinked by contact with at least one agent that formscrosslinks between collagen fibrils. Such a crosslinker may be selectedfrom one or more of an amine, carboxylic acid, sulfate, sulfite,sulfonate, aldehyde, hydrazide, sulfhydryl, diazirine, aryl, azide,acrylate, epoxide, phenol, chromium compound, vegetable tannin, andsyntan.

Crosslinking may be performed at a crosslinker concentration rangingfrom 1, 5, 10, 25, 50, 75 to 100 mM and may be conducted underconditions that uniformly expose collagen fibrils to the crosslinker sothat the average number of crosslinks formed is uniform and varies by nomore than 5, 10, 15, 20, 25, 30, 40, 45, or 50% in identical unitvolumes of the material.

A biofabricated material may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10% of a crosslinking agent based on the weight of the material orbased on the weight of the collagen or collagen fibrils in the material.The crosslinker may be present in a covalently or non-covalently form,for example, it may be covalently bound to the collagen fibrils. Acrosslinker may be uniformly present in the biofabricated material whereits concentration by weight (or by mole) varies by no more than 5, 10,15, 20, 25, 30, 40, 45, or 50% in identical unit volumes of thematerial.

The biofabricated material or biofabricated leather described abovecontains a lubricant. Not lubricated materials containing a network ofcollagen fibrils can be produced, such a precursor substrates for laterlubrication, but can lack the flexible and other useful properties of alubricated product. Lubricants may be incorporated in any amount thatfacilitates fibril movement or that confers leather-like properties suchas flexibility, decrease in brittleness, durability, strength, increaseresistance to fracture or tearing, or water resistance. A lubricantcontent can range from about 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60% by weight of thebiofabricated leather.

Lubricants used in embodiments of the invention include, but are notlimited to fats, biological, mineral or synthetic oils, cod oil,sulfonated oil, polymers, resins, organofunctional siloxanes, and otheragents used for fatliquoring conventional leather; mixtures thereof.Other lubricants include surfactants, anionic surfactants, cationicsurfactants, cationic polymeric surfactants, anionic polymericsurfactants, amphiphilic polymers, fatty acids, modified fatty acids,nonionic hydrophilic polymers, nonionic hydrophobic polymers, polyacrylic acids, poly methacrylic, acrylics, natural rubbers, syntheticrubbers, resins, amphiphilic anionic polymer and copolymers, amphiphiliccationic polymer and copolymers and mixtures thereof as well asemulsions or suspensions of these in water, alcohol, ketones, and othersolvents.

Solutions or emulsions containing a lubricant may be employed aslubricants, for examples, resins and other hydrophobic lubricants may beapplied as emulsions or in solvents suitable for dissolving them. Suchsolutions may contain any amount of the lubricant suitable forapplication to or incorporation into a biofabricated leather. Forexample, they may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 70, 80, 90, 95, or 99% of a lubricant or thesame, or a corresponding amount to volume of other ingredients, such asat least one aqueous solvent, such as water, alcohols, such C₁-C₆alcohols, like ethanol, ketones, such as C₁-C₆ ketones, aldehydes, suchas C₁-C₆ aldehydes, waxes, surfactants, dispersants or other agents.Lubricants may be in various forms, such as O/W or W/O emulsions, inaqueous or hydrophobic solutions, in sprayable form, or other formssuitable for incorporation or application to a biofabricated material.

Lubricants can be distributed uniformly throughout a biofabricatedmaterial such that the concentration of the lubricant in identical unitvolumes of the material varies by no more than 5, 10, 15, 20, 35, 30,40, or 50% and may be compounded or mixed into forms suitable foruniform application to or into a biofabricated material.

Some embodiments of a biofabricated product exhibit many advantageousproperties similar to leather or new or superior properties compared toconventional leather.

In other embodiments a biofabricated leather can have an elastic modulusof at least 100 kPA. It can range from 100 kPa to 1,000 MPa as well asany intermediate value in this range, such as 0.1, 0.2, 0.3 , 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 300,400, 500, 600, 700, 800, 900, or 1,000 MPA.

Some embodiments will exhibit a uniform elasticity, wherein the elasticmodulus varies by no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50%when measured at angles differing by 30, 60, or 90 degrees (or at otherangles) across identical lengths or widths (or volumes or fixedcross-sectional areas) of the material.

Some embodiments are stretchable and can be elongated by 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200,250 to 300% of its length in a relaxed state. This range includes allintermediate values.

In some embodiments, a biofabricated leather can have a tensile strengthof at least 1 kPA. It can range from 1 kPa to 100 MPa as well as anyintermediate value in this range, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,50, 100, 200, 300, 400, 500 kPA; 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80,90, or 100 MPa. Some embodiments will exhibit a uniform tensilestrength, wherein the tensile strength varies by no more than 5, 10, 15,20, 25, 30, 35, 40, 45, or 50% when measured at angles differing by 30,60, or 90 degrees (or at other angles) across identical lengths orwidths (or volumes or fixed cross-sectional areas) of the material.

Some biofabricated leathers exhibit tear strength or resistance of atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,100, 150 or 200% more than that of a conventional top grain or otherleather of the same thickness comprising the same type of collagen,e.g., bovine Type I or Type III collagen, processed using the samecrosslinker(s) or lubricants. . Some embodiments will exhibit a uniformtear resistance which varies by no more than 5, 10, 15, 20, 25, 30, 35,40, 45, or 50% when measured at angles differing by 30, 60, or 90degrees (or at other angles) across identical lengths or widths (orvolumes or fixed cross-sectional areas) of the material. A biofabricatedmaterial may have a tear strength ranging from about 1 to 500 N, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425,450, 475, or 500 as well as any intermediate tear strength within thisrange.

A biofabricated leather may have a softness as determined by ISO 17235of 2, 3, 4, 5, 6, 7, 8, 10, 11, 12 mm or more. v. Some embodiments willexhibit a uniform softness which varies by no more than 5, 10, 15, 20,25, 30, 35, 40, 45, 50, or 100% when measured in otherwise identicalunit areas or volumes of the biofabricated material.

In other embodiments, a biofabricated material exhibits a customizedthickness to provide top grain like products without the requirement forcorium backing. In some embodiments the material will have a top andbottom surface or an inner and outer surface which have identical orsubstantially the same grain, grain texture, feel, and appearance. Otherembodiments are embossed with a pattern, distressed, or printed, stainedor painted. Other embodiments have a surface coating or surface finish,which may be distributed uniformly on or throughout the material suchthat its concentration by weight in identical unit volumes or over unitareas of the material varies by no more than 5, 10, 15, 20, 25, 30, 35,40, 45, or 50%. Some embodiments may contain a dye, stain, resin,polymer, pigment or paint, optionally, wherein the dye, stain, resin,polymer, pigment or paint is distributed uniformly throughout thematerial such that its concentration by weight in identical unit volumesor on unit areas of the material varies by no more than 5, 10, 15, 20,25, 30, 35, 40, 45, or 50%.

Certain embodiments of the biofabricated material described above maycontain fillers as well as other substances or components incorporatedinto the network of collagen fibrils. For example, some embodiments willcontain a filler, such as at least one of polymeric microsphere(s),bead(s), fiber(s), wire(s), or organic salt(s). These can be selected soas to control the organization of the dehydrated collagen fibril networkby keeping the fibrils spaced apart during drying. A filler may besoluble under some conditions or otherwise in a form that permits itremoval from a biofabricated material after drying or other processing.

Other embodiments include at least one woven or nonwoven materialincorporated into the network of collagen fibrils or a network ofcollagen fibers incorporated into the nonwoven or woven material.

In some embodiments the biofabricated material will be incorporated intoother products such as footwear, clothing, sportswear, uniforms,wallets, watchbands, bracelets, luggage, upholstery, or furniture.

Embodiments of the Method of Making

The method according to the invention includes, but is not limited to,the following embodiments.

A method for making:

(i) a material comprising a network of non-human collagen fibrils,wherein less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or40% by weight of the collagen fibrils in the material are in the form ofcollagen fibers having a diameter of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μm ormore and/or are in the form of fibrils aligned for 25, 50, 100, 150,200, 250, 300, 350 or 400 μm or more of their lengths; wherein saidmaterial contains no more than 10, 15, 20, 25, 30, 35,40, 45, 50, 55 or60% by weight water; and wherein said material contains at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40% of a lubricant, comprising inany order: fibrillating an aqueous solution or suspension of non-humancollagen molecules into collagen fibrils, crosslinking said collagenfibrils by contacting them with at least one crosslinking agent,dehydrating the crosslinked collagen fibrils so that they contain lessthan 40% by weight water, incorporating at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, or 40% by weight of at least one lubricant into saidmaterial, and, optionally, casting, molding, or otherwise forming saidmaterial that comprises a top and bottom surface or an inner and outersurface; or

(ii) a material comprising a network of recombinant non-human collagenfibrils, wherein the collagen contains substantially no3-hydroxyproline, and, optionally, substantially no hydroxylysine;wherein said material contains no more than 10, 15, 20, 25, 30, 35,40,45, 50, 55 or 60% by weight water; and wherein said material contains atleast 1% of a lubricant comprising in any order: fibrillating an aqueoussolution or suspension of non-human collagen molecules into collagenfibrils,_rosslinking said collagen fibrils by contacting them with atleast one crosslinking agent,_dehydrating the crosslinked collagenfibrils so that they contain no more than 5, 10, 15, 20 or 25% by weightwater, and incorporating at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, or50% by weight of at least one lubricant into said material, and,optionally, casting, molding, or otherwise forming said material thatcomprises a top and bottom surface or an inner and outer surface.

The collagen or collagenous material for use in this method may comprisemammalian collagen, such as bovine Type I, Type III collagen or theother types and sources of collagens or collagenous proteins describedherein. It may be obtained from a mammal or other animal or, in someembodiments expressed recombinantly by Escherichia coli, Bacillussubtilis, or another bacterium; by Pichia, Saccharomyces, or anotheryeast or fungi; by a plant cell; by an insect cell or by a mammaliancell.

Collagen for use in the methods disclosed herein may also be isolatedfrom cells, such as those described above, that are cultured in vitro,such as from cultured mammalian or animal cells. Alternatively, collagenor collagenous proteins may be obtained by other means, such as bychemical synthesis. It may different in chemical structure from collagenobtained from a natural source, for example, if may contain a lowercontent of, or substantially no hydroxylysine or 3-hydroxyproline,glycosylated or crosslinked amino acid residues, or otherpost-translational modifications of a collagen amino acid sequence.Alternatively, it may contain a higher content of hydroxylated aminoacid residues, glycosylated residues, crosslinks or other chemicalmodifications.

Preferably a collagen will not contain hair, hair follicle(s), or fat(s)of an animal that naturally expresses the collagen molecules it containsas these can detract from its uniformity, strength and aestheticproperties. For example, it may contain less than 1, 2, 3, 4, 5, 6, 7,8, 9, or 10% by weight of actin, keratin, elastin, fibrin, albumin,globulin, mucin, mucinoids, noncollagen structural proteins, and/ornoncollagen nonstructural proteins found in conventional leather. It maybe substantially free of other collagenous proteins, carbohydrates,nucleic acids, or lipids, or immunogens, antigens, or allergens found ina conventional leather, such as an animal that naturally expresses thecollagen molecules in a biofabricated material.

In some embodiments a collagen or collagen-like material for use in themethods described herein may be purified to substantial homogeneity ormay have a degree of purity not inconsistent with its ability to formfibrils, for example, it may contain 25, 30, 40, 50, 60, 70, 80, 90, 95or 99% by weight collagen based on its total protein content or based onits total weight. Mixtures of different types of collagen or collagensfrom different biological sources may be used in certain embodiments tobalance the chemical and physical properties of collagen fibrils or toproduce a mixture of fibrils having complementary properties. Suchmixtures may contain 1, 5, 10, 25, 50, 75, 95, or 99% by weight of afirst collagen and 99, 95, 90, 75, 50, 25, 10 or 1% by weight of asecond, third, or subsequent collagen component. These ranges includeall intermediate values and ratios of collagens where the total collagencontent of all collagen components by weight is 100%.

The methods disclosed herein can provide a biofabricated product havingsubstantially uniformly distributed fibrils, crosslinked fibrils,dehydrated fibrils and/or lubricated fibrils. For example, the fibrilsmay be distributed throughout the material so that the concentration byweight (or by number or average numbers) of the collagen fibrils inidentical unit volumes of the material varies by no more than 5, 10, 15,20, 25, 30, 35, 40, 45, or 50%.

In some embodiments the biofabricated material will be staking thematerial after the crosslinking, dehydrating and/or lubricating.

In the embodiments described herein, a collagen solution or suspensionis fibrillated, for example, by adjusting a salt concentration of thesolution or suspension, by adjusting its pH, for example, raising the pHof an acidic solution of collagen, or both. In some embodiments,fibrillation may be facilitated by including a nucleating agent. Saltsused for fibrillation include but are not limited to phosphate salts andchloride salts, such as Na₃PO₄, K₃PO₄, KCl, and NaCl. Salt concentrationduring fibrillation may be adjusted to range from 10 mM to 2M, or pH maybe adjusted to pH 5.5, 6.0, 6.5, 7.0, 8.0 or more with an acid, a base,or a buffer. Salt concentration and pH may be simultaneously adjusted toinduce or promote fibrillation. In certain embodiments of the methodsdescribed herein an aqueous solution or suspension of collagen moleculeshaving a pH below pH 6.0 can be fibrillated by adjusting the pH to pH6.0 to 8.0.

In some embodiments of the methods described herein, the collagenfibrils will be crosslinked during a process of their formation or aftercompletion of fibrillation.

In some embodiments, collagen fibrils are crosslinked by contacting themwith at least one amine, carboxylic acid, sulfate, sulfite, sulfonate,aldehyde, hydrazide, sulfhydryl, diazirine, aryl, azide, acrylate,epoxide, phenol, chromium compound, vegetable tannin, and syntan.

One or more crosslinkers may be added at a concentration ranging from 1mM to 100 mM, for example at a concentration of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 6, 70, 75, 80,85, 90, 95or 100 mM.

The time, temperature and other chemical and physical conditions ofcrosslinking may be selected to provide a particular degree ofcrosslinking among the collagen fibrils so that the resultingcrosslinked fibrils contain a particular degree of one or more differentcrosslinkages. A resulting crosslinked fibril preparation may contain atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% or more of a crosslinking agentbased on the weight of the crosslinking agent and the weight of thecollagen or on the weight of a crosslinked network of collagen fibrils,such as a hydrogel. The crosslinker may be covalently- or non-covalentlybound to the collagen fibrils. The numbers of crosslinks between oramong collagen molecules, tropocollagen, or fibrils in identical unitvolumes of the material after crosslinking, or an average number ofcrosslinks between collagen molecules, tropocollagen, or collagenfibrils, may vary by no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, or50%.

The methods described herein require a dehydration or dewatering stepwhich may occur during fibrillation or crosslinking, or both, or afterfibrillation and crosslinking are substantially complete.

In some embodiments, dehydrating involves contacting a network ofcollagen fibrils with acetone, syntan, or other agent that removes boundwater from collagen. In other embodiments, some water may be removedfrom a fibril preparation or crosslinked fibril preparation byfiltration or evaporation and water remaining associated with thenetwork of collagen fibrils then removed using a solvent such as acetoneor other chemical agents that remove water.

The methods described herein generally require lubrication of thenetwork of collagen fibrils produced. Lubrication may take place duringfibrillation, crosslinking, of dehydration, or during any of thesesteps, or after one or more of these steps is substantially complete.

In some embodiments lubrication will involve contacting a network ofcrosslinked collagen fibrils with one or more lubricants such as fats,biological, mineral or synthetic oils, cod oil, sulfonated oil,polymers, organofunctional siloxanes, and other agent used forfatliquoring conventional leather; or mixtures thereof.

In other embodiments, lubricant(s) will be applied using methods thatfacilitate uniform lubrication of a dehydrated crosslinked network ofcollagen fibrils, so that the concentration of the lubricant by weightin identical unit volumes of the material varies by no more than 5, 10,15, 20, 25, 30, 35, 40, 45, or 50%. Such application may occur bydip-coating, spray-coating, vapor deposition, spin-coating, Doctor Bladecoating, brush coating as well as other known coating or depositionmethods.

In further embodiments of the methods described herein, a surfacecoating or surface finish is applied to a biofabricated material. Whilethese may be applied to a surface of a material comprising a network ofcollagen fibrils during the various steps of the preparation of abiofabricated material, they will generally be applied to a crosslinked,dehydrated and lubricated product. The uniform lubrication made possibleby the methods described herein facilitate the successful uniformapplication and adherence of such coatings or finishes.

In other embodiments, the methods described herein can includeincorporating or contacting a biofabricated material during the varioussteps of its preparation or after it has been crosslinked, dehydratedand lubricated with other functional ingredients including, but notlimited to a dye, stain, pigment, resin, polymer, or paint. In furtherembodiments, these functional ingredients may be applied or incorporatedunder conditions that uniformly distribute these agents on or throughoutthe material so that their concentration by weight in identical unitvolumes of the material varies by no more than 5, 10, 15, 20, 25, 30,35, 40, 45, or 50%.

In other embodiments, the method described herein involves incorporatinga filler into a biofabricated material during the various steps of itspreparation or after it has been crosslinked, dehydrated and lubricated.Generally, these fillers are incorporated prior to dehydration, forexample, during fibrillation or crosslinking. Such fillers include, butare not limited to polymeric microspheres, beads, fibers, wires, ororganic salts.

Some embodiments of the methods described above will involveincorporating into or onto a biofabricated material during or after itspreparation at least one woven or nonwoven material. For example, byfiltering crosslinked fibrils using a woven or nonwoven paper or fabricmaterial. Other embodiments involve incorporating a biofabricatedmaterial during or after its preparation into at least one woven ornonwoven material.

Commercial embodiments of the method involving incorporating abiofabricated material into products such as footwear, clothing,sportswear, uniforms, wallets, watchbands, bracelets, luggage,upholstery, furniture, or other industrial, commercial or consumerproducts.

The following non-limiting Examples are illustrative of the presentinvention. The scope of the invention is not limited to the detailsdescribed in these Examples.

EXAMPLE 1 Controlling the Thickness of Biofabricated Leather

Hydrogels of extacted bovine type I collagen were formed at differentcollagen concentrations and volumes to produce dried collagen materialsof different thicknesses. Collagen was dissolved in 0.01N HCl at either5 g/L or 9 g/L, then 1 part 10× PBS was added to 9 parts dissolvedcollagen to induce collagen fibrillation and gel formation.

Solutions of either 0.8 L or 1.6 L of the fibrillating collagen werethen cast into molds and incubated at 25° C. to allow hydrogelformation. The 0.8 L solution produced a gel of 1.5 cm thickness whilethe 1. 7 L solution produced a gel of 3.0 cm thickness. These gels weredehydrated and lubricated in acetone, then dried and mechanically stakedinto a leather like material. The thickness of the final dried materialcorrelated with the total amount of collagen in the starting hydrogel.

The thickness of biofabricated leather was controlled by varying itstotal collagen content. Samples A, B and C were produced using 4, 7.2 or14.4 gr of collagen, respectively, in a volume (hydrated gel area) of525 cm². Biofabricated leathers were produced from each sample bycrosslinking, lubricating and dewatering As shown in Table 1, increasingthe content of collagen in the gels increased the thickness of theresulting biofabricated leather.

TABLE 1 Gel Gel Leather Density Gel Volume Thickness Total ThicknessSample (g/L) (L) (cm) Collagen (g) (mm) A 5 0.8 1.5 4 0.1 B 9 0.8 1.57.2 0.2 C 9 1.6 3.0 14.4 1.1

EXAMPLE 2 Production of Biofabricated Leather from Type I Collagen

Type I collagen was purchased from Wuxi Biot Bio-technology Company,ltd. (Medical Collagen Sponge). The collagen was isolated from bovinetendon by acid treatment followed by pepsin digestion, and was purifiedby size exclusion chromatography, frozen and lyophilized.

The lyophilized protein (4.1 g) was dissolved in 733 ml 0.01 N HCL usingan overhead mixer. After the collagen was adequately dissolved, asevidenced by a lack of solid collagen sponge in the solution (at leastlhr mixing at 1600 rpm), 82 uL of the tanning agent Relugan GTW wasadded to the solution followed by 81 mL of a 10× PBS, pH 11.2 to raisethe pH of the solution to 7.2.

The solution was then mixed for 3 min before pouring the solution into asilicon mold. The collagen solution was incubated in the silicon moldfor 2 hrs at 25° C. to allow the collagen to fibrillate into aviscoelastic hydrogel.

Plateau of rheological properties along with solution opacity (asmeasured by absorbance of 425 nm light) indicated that fibrillation wascomplete at this point and the presence of collagen fibrils wasconfirmed with scanning electron microscopy (FIG. 3) and transmissionelectron microscopy (FIG. 4).

The fibrillated collagen hydrogel was removed from the molds and placedin 700 mL of acetone in a plastic jar and shaken on an orbital shaker at40 rpm at 25° C. The hydrogel was dehydrated by refreshing the acetoneafter an overnight incubation followed by 5× 1 hr washes and anotherovernight incubation. Acetone was refreshed after each wash to removewater from the gel.

Following acetone dehydration, the collagen gel was incubated in a fatliquor solution containing 20% (v/v) of either cod liver oil or castoroil in 80% acetone or ethanol, respectively, overnight while shaking at40 rpm.

Following incubation in the fat liquor solution, the collagen gel wasdried at 37 C. After drying, the material became soft and leather-likeor a biofabricated leather. Excess oil can be removed to improve theleather-like aesthetic of the materials.

The biofabricated leather had a grain texture on both the top and bottomsurfaces and consistently absorbed dyes on both the top and bottomsurfaces.

EXAMPLE 3 Production of Biofabricated Leather from Type III Collagen

A solution of recombinant collagen type III at 2.5 mg/ml in 0.01 N HCl(FibroGen, Inc.) was fibrillated by adding 1 part of a 200 mM of sodiumphosphate solution (22 mL), pH 11.2 to 9 parts of the collagen solution(200 mL) to increase the pH to 7 and stirred 2 hours at roomtemperature.

Fibrillation was confirmed by measuring 400nm absorbance of the solutionover time.

After fibrillation, the fibrils were tanned by adding Relugan GTW (2%w/w offer on the collagen) to the fibril suspension and mixing for 30min.

The tanned collagen fibrils were then centrifuged at 3,500 RPM for 30minutes to concentrate the fibrils to a concentration of 10 mg/ml . The10 mg/ml fibril pellet was further centrifuged using an ultra-centrifugeat 21,000 RPM for 30 minutes yielding a fibril gel with a concentrationof ˜40-50 mg/ml .

The physical properties of the fibril gel were assessed with arheometer.

The storage modulus and complex viscosity demonstrate a mostly elasticmaterial.

This fibril gel was then dried in a food dehydrator set to 37° C. for 18hrs.

After drying, the material was dyed and retanned by incubating in asolution of Lowepel acid black dye (2% w/w offer on the collagen) andLubritan WP (20% w/w offer on the collagen).

The material was drummed in this solution and squeezed to ensurepenetration of dye and syntan into the material. The material was thenfinally dried and staked to produce a leather-like material.

EXAMPLE 4 Production of Biofabricated Leather from Type III Collagen

Recombinant collagen type III was purchased from Fibrogen, Inc. Thecollagen was supplied at a concentration of 2.5 mg/mL in 0.01N HCl.

To initiate the assembly of collagen fibrils, 1 part 200 mM Na₂HPO₄, pH11.2 (100mL) was added to 9 parts of the stock collagen type IIIsolution at room temperature to bring the solution to pH 7.2. Thesolution was mixed at 1600 rpm for lhr using an overhead mixer.

After 1 hr of stirring, the collagen fibrils were reacted with ReluganGTW which was added to the solution at a 2% (w/w) offer on the mass ofthe collagen. The solution was mixed at 1600 rpm for lhr using anoverhead mixer.

Lipoderm Al and Tanigan FT were then added to the solution at offers of80% (w/w) each on the mass of the collagen. The solution was mixed at1600 rpm for 30 min using an overhead mixer. The pH of the solution wasthen lowered to 4 using a 10% (v/v) formic acid solution. The solutionwas mixed at 1600 rpm for 30 min using an overhead mixer.

144 mL of the solution was then filtered through a 47 mm Whatman no. 1membrane using a Buchner funnel attached to a vacuum pump (pressure of-27 in Hg) and a rubber dam on top of the Buchner funnel. Vacuum waspulled for 18 hrs.

The concentrated fibril tissue was then allowed to dry under ambientconditions and hand staked for 30 min by rolling, bending and pullingthe material to produce a leather-like material.

EXAMPLE 5 Expancel

Type I bovine collagen, isolated from bovine tendon by acid treatmentfollowed by pepsin digestion and purified by size exclusionchromatography, frozen and lyophilized, was purchased from Wuxi BiotBio-technology co., Ltd. (Medical Collagen Sponge).

Using an overhead mixer, 10 gr of the lyophilized collagen protein wasdissolved by mixing at 1,600 rpm in 1 L of 0.01N HCl, pH 2, for at leastone hour until no solid collagen sponge was present.

111.1 ml of 200 mM sodium phosphate (pH adjusted to 11.2 with sodiumhydroxide) was then added to raise the pH of the collagen solution to7.2.

The pH 7.2 collagen solution was then stirred for 10 minutes and 0.1 mlof a 20% Relugan GTW (BASF) as a crosslinker, which was 2% on the weightof collagen, was added to produce crosslinked collagen fibrils.

The crosslinked collagen fibrils were then mixed with 5 ml of 20%Tanigan FT (Lanxess) and stirred for one hour.

Subsequently, 1 gr of Expancel Microspheres 461 WE 20 d36 (AkzoNobel),which is 10% of the weight of the collagen) and 40 ml of Truposol Ben(Trumpler), which is 80% of the weight of the collagen, were added andstirred for an additional our using an overhead stirrer.

The pH of the solution was the reduced to pH 4.0 by addition of 10%formic acid and stirred for an hour.

After the reduction in pH, 150 ml of the solution was filtered through90 mm Whatman No.1 membrane using a Buchner funnel attached to a vacuumpump at a pressure of −27 mmHg.

The concentrated fibril tissue was then allowed to dry under ambientconditions and hand staked for 30 minutes by rolling, bending andpulling the material to produce a leather-like material.

EXAMPLE 6 Titanium Dioxide (White Pigment)

Type I bovine collagen was purchased from Wuxi Biot Bio-technology co.,Ltd. (Medical Collagen Sponge). This source of collagen is type Icollagen isolated from bovine tendon by acid treatment followed bypepsin digestion and purified by size exclusion chromatography, frozenand lyophilized. The lyophilized protein (10 grams) was dissolved in 1 Lof 0.01N HCl, pH 2 using an overhead mixer. After the collagen wasadequately dissolved, as evidenced by a lack of solid collagen sponge inthe solution (at least 1 hr mixing at 1,600 rpm), 111.1 ml of 200millimolar sodium phosphate (pH adjusted to 11.2 with sodium hydroxide)to raise the pH of the solution to 7.2. The resulting collagen solutionwas stirred for 10 minutes and 0.1 ml of a 20% R.elugan GTW (BASF)crosslinker solution, which was 2% on the weight of collagen.

To the crosslinked collagen fibril solution was added 5 mis of 0%Tanigan FT (Lanxess) was added followed by stirring for one hour.

Following Tanigan-FT addition, 1 gr Expancel Microspheres (10% on theweight of collagen) 461 WE 20 d36 (AkzoNobel) ,40 mls (80% on the weightof collagen) of Truposol Ben (Trampler) and 2 mls (10% on the weight ofcollagen) of PPE White HS a pa (Stahl) was added and stirred foradditional hour using an overhead stirrer.

The pH of the solution was reduced to 4.0 using 10% formic acid andstirred for an hour.

After pH change, 150 ml of the solution was filtered through 90 mMWhatman No.1 membrane using a Buchner funnel attached to a vacuum pumpat a pressure of −27 mmHg.

The concentrated fibril tissue was then allowed to dry under ambientconditions and hand staked for 30 minutes by rolling, bending andpulling the material to produce a leather-like material.

EXAMPLE 7 Hycar Resin (26552)

Bovine collagen was purchased from Wuxi Biot Bio-technology co., Ltd.(Medical Collagen Sponge). This source of collagen is type I collagenisolated from bovine tendon by acid treatment followed by pepsindigestion and purified by size exclusion chromatography, frozen andlyophilized.

The lyophilized protein (10 grams) was dissolved in 1 litre of 0.01NHCl, pH 2 using an overhead mixer. After the collagen was adequatelydissolved, as evidenced by a lack of solid collagen sponge in thesolution (at least 1 hr mixing at 1600 rpm), 111.1 ml of 200 mM sodiumphosphate (pH adjusted to 11.2 with sodium hydroxide) to raise the pH ofthe solution to 7.2.

The resulting collagen solution was stirred for 10 minutes and 0.1 ml ofa 20% Relugan GTW (BASF) crosslinker solution which was 2% of the weightof the collagen, tanning agent solution was added.

To the crosslinked collagen fibril solution was added 5 mls of 20%Tanigan FT (Lanxess) was added and stirred for one hour. FollowingTanigan-FT addition, 1 gram Expancel Microspheres (10% on the weight ofcollagen) 461 WE 20 d36 (AkzoNobel), 40 mls (80% on the weight ofcollagen) of Truposol Ben (Trumpler) and 2 mls (10% on the weight ofcollagen) of PPE White HS a pa (Stahl) was added and added and stirredfor additional hour using an overhead stirrer.

The pH of the solution was reduced to 4.0 using 10% formic acid and avariety of offers of Hycar Resin 26552 (Lubrizol) was added and stirredfor an additional hour. Following pH change and resin addition 150 ml ofthe solution was filtered through 90 millimeter Whatman No.1 membraneusing a Buchner funnel attached to a vacuum pump at a pressure of −27mmHg. To facilitate activation, the Hycar Resin 26552 is mixed with thefibril solution and heated at 50° C. for 2 hrs.

The concentrated fibril tissue was then allowed to dry under ambientconditions and hand staked for 30 minutes by rolling, bending andpulling the material to produce a leather-like material. The addition ofresin lead to improved mechanical properties as shown below in FIG. 1.

Example Substrates Crosslinker Dehydrater Lubricant Result 5 Type Icollagen + Relugan Tanigan Truposol Leather- Expancel GTW FT likemicrospheres material 6 Type I collagen + Relugan Tanigan ″ Leather-Expancel GTW FT like microspheres material 7 Type I collagen + ReluganTanigan ″ Leather- Expancel GTW FT like microspheres material, bettermechanical properties

Relugan is a retanning agent based on polymer, resin or aldehyde.Tanigan is a sulfone-based syntan, Truposol Ben is a fatliquor forchrome-free leather. Lipoderm Liquor A1 is a fatliquor based on longchain alcohol, paraffin, anionic surfactants, in water Hycar Resin 26552formal dehyde-free acrylic based emulsion

Interpretation of Description

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.For example, as used herein, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. It will be further understood that the terms“comprises” and/or “comprising,” when used in this specification,specify the presence of stated features, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, steps, operations, elements, components, and/orgroups thereof. As used herein, the term “and/or” includes any and allcombinations of one or more of the associated listed items and may beabbreviated as “/”.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if a device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of over and under. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present invention.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising” means various components can be co-jointlyemployed in the methods and articles (e.g., compositions and apparatusesincluding device and methods). For example, the term “comprising” willbe understood to imply the inclusion of any stated elements or steps butnot the exclusion of any other elements or steps.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “substantially”, “about” or“approximately,” even if the term does not expressly appear. The terms“substantially”, “substantially no”, “substantially free”, “about” or“approximately” may be used when describing magnitude and/or position toindicate that the value and/or position described is within a reasonableexpected range of values and/or positions. For example, a numeric valuemay have a value that is +/−0.1% of the stated value (or range ofvalues), +/−1% of the stated value (or range of values), +/−2% of thestated value (or range of values), +/−5% of the stated value (or rangeof values), +/−10% of the stated value (or range of values), etc. Anynumerical range recited herein is intended to include all sub-rangessubsumed therein.

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

Although various illustrative embodiments are described above, any of anumber of changes may be made to various embodiments without departingfrom the scope of the invention as described by the claims. For example,the order in which various described method steps are performed mayoften be changed in alternative embodiments, and in other alternativeembodiments one or more method steps may be skipped altogether. Optionalfeatures of various device and system embodiments may be included insome embodiments and not in others. Therefore, the foregoing descriptionis provided primarily for exemplary purposes and should not beinterpreted to limit the scope of the invention as it is set forth inthe claims.

The examples and illustrations included herein show, by way ofillustration and not of limitation, specific embodiments in which thesubject matter may be practiced. As mentioned, other embodiments may beutilized and derived there from, such that structural and logicalsubstitutions and changes may be made without departing from the scopeof this disclosure. Such embodiments of the inventive subject matter maybe referred to herein individually or collectively by the term“invention” merely for convenience and without intending to voluntarilylimit the scope of this application to any single invention or inventiveconcept, if more than one is, in fact, disclosed. Thus, althoughspecific embodiments have been illustrated and described herein, anyarrangement calculated to achieve the same purpose may be substitutedfor the specific embodiments shown. This disclosure is intended to coverany and all adaptations or variations of various embodiments.Combinations of the above embodiments, and other embodiments notspecifically described herein, will be apparent to those of skill in theart upon reviewing the above description.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference,especially referenced is disclosure appearing in the same sentence,paragraph, page or section of the specification in which theincorporation by reference appears.

1. A method for making: (i) a material comprising a network of non-humancollagen fibrils, wherein less than 10% by weight of the collagenfibrils in the material are in the form of collagen fibers having adiameter of 5 μm or more, are in the form of fibrils aligned for 100 μmor more of their lengths, or both; wherein said material contains nomore than 40% by weight water; and wherein said material contains atleast 1% of a lubricant, comprising in any order: fibrillating anaqueous solution or suspension of non-human collagen molecules intocollagen fibrils, crosslinking said collagen fibrils by contacting themwith at least one crosslinking agent, dehydrating the crosslinkedcollagen fibrils so that they contain less than 40% by weight water,incorporating at least 1% by weight of at least one lubricant into saidmaterial; or (ii) a material comprising a network of recombinantnon-human collagen fibrils, wherein the collagen contains substantiallyno 3-hydroxyproline; wherein said material contains no more than 25% byweight water; and wherein said material contains at least 1% of alubricant comprising in any order: fibrillating an aqueous solution orsuspension of non-human collagen molecules into collagen fibrils,crosslinking said collagen fibrils by contacting them with at least onecrosslinking agent, dehydrating the crosslinked collagen fibrils so thatthey contain no more than 25% by weight water, and incorporating atleast 1% by weight of at least one lubricant into said material.
 2. Themethod of claim 1, wherein said method is (i).
 3. The method of claim 2,wherein the non-human collagen molecules are isolated from a mammal orother animal.
 4. The method of claim 2, wherein the non-human collagenmolecules are recombinant non-human collagen molecules that containsubstantially no 3-hydroxyproline.
 5. The method of claim 1, whereinsaid method is (ii).
 6. The method of claim 5, wherein less than 10% byweight of the collagen fibrils in the material is in the form ofcollagen fibers having a diameter of 5 μm or more, is in the form offibrils aligned for 100 μm or more of their lengths, or both.
 7. Themethod of claim 1, further comprising casting, molding or forming thematerial into one having a top and bottom surface or an inner and outersurface.
 8. The method of claim 1, wherein fibrillating comprises addinga salt or a combination of salts.
 9. The method of claim 1, whereinfibrillating comprises adjusting the pH of the aqueous solution orsuspension of collagen to a pH of 5.5 or more with an acid, a base, or abuffer.
 10. The method of claim 1, wherein said crosslinker is at leastone selected from the group consisting of an amine, carboxylic acid,sulfate, sulfite, sulfonate, aldehyde, hydrazide, sulfhydryl, diazirine,aryl, azide, acrylate, epoxide, phenol, chromium compound, vegetabletannin, and syntan.
 11. The method of claim 1 that comprises dehydratingthe network of collagen fibrils by contacting them with at least onedehydrating or dewatering solvent.
 12. The method according to claim 1,further comprising uniformly applying the at least one lubricant on orthroughout the material, wherein the concentration by weight of the atleast one lubricant on or in identical unit volumes of the materialvaries by no more than 20%.
 13. The method according to claim 1, furthercomprising uniformly applying a surface coating or surface finish on orthroughout the material, wherein the concentration by weight of thesurface coating or surface finish on or in identical unit volumes of thematerial varies by no more than 20%.
 14. The method according to claim1, further comprising uniformly distributing a dye, stain, pigment,resin, polymer, or paint on or throughout the material, wherein theconcentration by weight of dye, stain, pigment, resin, polymer, or paintin identical unit volumes of the material varies by no more than 20%.15. The method according to claim 1, further comprising incorporatinginto or onto said material at least one filler; or further comprisingincorporating into or onto said material at least one woven or nonwovenmaterial or incorporating said material into at least one woven ornonwoven material.